Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples

ABSTRACT

The technology described herein generally relates to systems for extracting polynucleotides from multiple samples, particularly from biological samples, and additionally to systems that subsequently amplify and detect the extracted polynucleotides. The technology more particularly relates to microfluidic systems that carry out PCR on multiple samples of nucleotides of interest within microfluidic channels, and detect those nucleotides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/124,672, filed Sep. 7, 2018, which is a continuation of U.S. patentapplication Ser. No. 14/941,087, filed Nov. 13, 2015 and issued as U.S.Pat. No. 10,071,376 on Sep. 11, 2018, which is a continuation of U.S.patent application Ser. No. 12/218,498, filed Jul. 14, 2008 and issuedas U.S. Pat. No. 9,186,677 on Nov. 17, 2015, which claims the benefit ofpriority under 35 U.S.C. § 119(e) to U.S. Provisional Application No.60/959,437, filed Jul. 13, 2007, and is a continuation-in-part of U.S.patent application Ser. No. 11/985,577, filed Nov. 14, 2007 and issuedon Aug. 16, 2011 as U.S. Pat. No. 7,998,708. The disclosures of all ofthe above-referenced prior applications, publications, and patents areconsidered part of the disclosure of this application, and areincorporated by reference herein in their entirety.

TECHNICAL FIELD

The technology described herein generally relates to systems forextracting polynucleotides from multiple samples, particularly frombiological samples, and additionally to systems that subsequentlyamplify and detect the extracted polynucleotides. The technology moreparticularly relates to microfluidic systems that carry out PCR onmultiple samples of nucleotides of interest within microfluidicchannels, and detect those nucleotides.

BACKGROUND

The medical diagnostics industry is a critical element of today'shealthcare infrastructure. At present, however, in vitro diagnosticanalyses no matter how routine have become a bottleneck in patient care.There are several reasons for this. First, many diagnostic analyses canonly be done with highly specialist equipment that is both expensive andonly operable by trained clinicians. Such equipment is found in only afew locations—often just one in any given urban area. This means thatmost hospitals are required to send out samples for analyses to theselocations, thereby incurring shipping costs and transportation delays,and possibly even sample loss or mishandling. Second, the equipment inquestion is typically not available ‘on-demand’ but instead runs inbatches, thereby delaying the processing time for many samples becausethey must wait for a machine to fill up before they can be run.

Understanding that sample flow breaks down into several key steps, itwould be desirable to consider ways to automate as many of these aspossible. For example, a biological sample, once extracted from apatient, must be put in a form suitable for a processing regime thattypically involves using PCR to amplify a vector (such as a nucleotide)of interest. Once amplified, the presence of a nucleotide of interestfrom the sample needs to be determined unambiguously. Preparing samplesfor PCR is currently a time-consuming and labor intensive step, thoughnot one requiring specialist skills, and could usefully be automated. Bycontrast, steps such as PCR and nucleotide detection (or ‘nucleic acidtesting’) have customarily only been within the compass of speciallytrained individuals having access to specialist equipment.

There is a need for a method and apparatus of carrying out samplepreparation on samples in parallel, with or without PCR and detection onthe prepared biological samples, and preferably with high throughput,but in a manner that can be done routinely at the point of care, orwithout needing the sample to be sent out to a specialized facility.

The discussion of the background herein is included to explain thecontext of the inventions described herein. This is not to be taken asan admission that any of the material referred to was published, known,or part of the common general knowledge as at the priority date of anyof the claims.

Throughout the description and claims of the specification the word“comprise” and variations thereof, such as “comprising” and “comprises”,is not intended to exclude other additives, components, integers orsteps.

SUMMARY

A diagnostic apparatus, comprising: a first module configured to extractnucleic acid simultaneously from a plurality of nucleic-acid containingsamples, wherein the first module comprises one or more racks, eachconfigured to accept a number of samples and a corresponding number ofholders, wherein each holder comprises a process chamber, a wastechamber, one or more pipette tips, and one or more receptacles, whereinthe one or more receptacles contain respectively sufficient quantitiesof one or more reagents for carrying out extraction of nucleic acid froma sample; a magnetic separator configured to move relative to theprocess chambers of each holder; a heater assembly configured toindependently heat each of the process chambers; and a liquid dispenserconfigured to carry out fluid transfer operations on two or more holderssimultaneously; and a second module configured to simultaneously amplifythe nucleic acid extracted from the plurality of samples, wherein thesecond module comprises: one or more bays, each configured to receive amicrofluidic cartridge, wherein the cartridge is configured toseparately accept and to separately amplify the nucleic acid extractedfrom multiple samples; and one or more detection systems.

A diagnostic apparatus comprising: one or more racks, on each of whichis mounted a number of nucleic acid containing samples and acorresponding number of holders, wherein each holder comprises a processchamber, a waste chamber, one or more pipette tips, and one or mornreceptacles, wherein the one or more receptacles contain, respectively,sufficient quantities of one or more reagents for carrying outextraction of nucleic acid from a sample; a magnetic separator movablefrom a first position to a second position adjacent to the processchamber of each of the one or more holders; a heater assembly comprisinga number of heater units, each of which is in thermal contact with oneof the process chambers; one or more bays, each bay having a shapecomplementary to a shape of a microfluidic cartridge, wherein thecartridge comprises a number of inlets each of which is in fluidcommunication with one of a number of channels in which nucleic acidextracted from one of the number of samples is amplified, and whereinthe cartridge further comprises one or more windows that permitdetection of amplified nucleic acid; a liquid dispenser having one ormore dispensing heads, wherein the liquid dispenser is movable from afirst position above a first holder to a second position above a secondholder, and is movable from the first position above the first holder toa different position above the first holder, and is further movable froma position above one of the holders to a position above one of thenumber of inlets; and one or more detection systems positioned inproximity to the one or more windows.

A diagnostic instrument comprising: a liquid handling unit that extractsnucleic acid from a sample in a unitized reagent strip; a microfluidiccartridge that, in conjunction with a halter element, carries outreal-time PCR on nucleic acid extracted from the sample; and a detectorthat provides a user with a diagnosis of whether the sample contains anucleotide of interest.

Also described herein are methods of using the diagnostic apparatus,including a method of diagnosing a number of samples in parallel, usingthe apparatus.

A unitized reagent holder, comprising: a strip, to which is attached: asingle process tube; one or more receptacles, each of which holding areagent selected from the group consisting of: a sample preparationreagent, PCR reagents for a first analyte, and one or more liquidreagents; a waste tube; one or more sockets configured to hold one ormore pipette tips; and a pipette tip sheath configured to surround theone or more pipette tips.

A liquid dispenser, comprising: one or more sensors; a manifold; one ormore pumps in fluid communication with the manifold; one or moredispense heads in fluid communication with the manifold; a gantry thatprovides freedom of translational motion in three dimensions; andelectrical connections that accept electrical signals from an externalcontroller, wherein the liquid dispenser has no inlet or outlet forfluids, other than through the one or more pumps.

A separator for magnetic panicles, comprising: one or more magnetsaligned linearly, a motorized shaft upon which the one or more magnetscan rise or fall in such a manner that the one or more magnets attainsclose proximity to one or more receptacles containing magneticparticles; and control circuitry to control motion of the motorizedshaft.

An integrated separator and heater, comprising a heater assembly,wherein the heater assembly comprises a plurality of independentlycontrollable heater units, each of which is configured to accept and toheat a process chamber, one or more magnets aligned linearly; amotorized shat upon which the one or more magnets can rise or fall insuch a manner that the one or more magnets attains close proximity toone or more of the process chambers; and control circuitry to controlmotion of the motorized shaft and heating of the heater units.

A preparatory apparatus comprising: a first module configured to extractnucleic acid simultaneously from a number of nucleic-acid containingsamples, wherein the first module comprises: one or more racks, eachconfigured to accept the number of samples and a corresponding number ofholders, wherein each holder comprises a process chamber, a wastechamber, one or more pipette tips, and one or more receptacles, whereinthe one or more receptacles contain, respectively, sufficient quantitiesof one or more reagents for carrying out extraction of nucleic acid froma sample; a magnetic separator configured to move relative to theprocess chambers of each holder; a heater assembly configured toindependently heat each of the process chambers; and a liquid dispenserconfigured to carry out fluid transfer operations on two or more holderssimultaneously; and a second module configured to receive and to storethe nucleic acid extracted from the number of samples.

A preparatory apparatus comprising: one or more racks, on each of whichis mounted a number of nucleic acid containing samples and acorresponding number of holders, wherein each holder comprises a processchamber, a waste chamber, one or more pipette tips, and one or morereceptacles, wherein the one or more receptacles contain, respectively,sufficient quantities of one or more reagents for carrying outextraction of nucleic acid from a sample; a magnetic separator movablefrom a first position to a second position adjacent to the processchambers of each holder, a heater assembly comprising a number of heaterunits, each of which is in contact with a process chamber; a liquiddispenser movable from a first position above a first holder to a secondposition above a second holder; and a storage compartment having anumber of compartments, wherein each compartment stores the nucleic acidextracted from one of the number of samples.

A unitized reagent holder, comprising: a strip, to which is attached: asingle process tube; one or more receptacles, each of which holding areagent selected from the group consisting of: a sample preparationreagent, and one or more liquid reagents; a waste tube; one or moresockets configured to hold one or more pipette tips; and a pipette tipsheath configured to surround the one or more pipette tips.

The present technology additionally includes a process for extractingnucleic acid from multiple samples in parallel, using the apparatus asdescribed herein.

BRIEF DESCRIPTION OF SELECTED DRAWINGS

FIG. 1A shows a schematic of a preparatory apparatus; FIG. 1B shows aschematic of a diagnostic apparatus.

FIG. 2 shows a schematic of control circuitry.

FIGS. 3A and 3B show exterior views of an exemplary apparatus.

FIG. 4 shows an exemplary interior view of an apparatus.

FIG. 5 shows perspective views of an exemplary rack for sample holders.

FIG. 6 shows perspective views of the rack of FIG. 5 in conjunction witha heater unit.

FIG. 7 shows a perspective view of an exemplary rack for sample holders.

FIGS. 8A-8K show various views of the rack of FIG. 7.

FIG. 9 shows an area of an apparatus configured to accept a rack of FIG.7.

FIGS. 10A and 10B show an first exemplary embodiment of a reagent holderhaving a pipette sheath, in perspective view (FIG. 10A) and undersideview (FIG. 10B).

FIG. 11 shows an exemplary embodiment of a reagent holder not having apipette sheath, in perspective view.

FIGS. 12A-12C show a second exemplary embodiment of a reagent holderhaving a pipette sheath, in perspective view (FIG. 12A) andcross-sectional view (FIG. 12B), and exploded view (FIG. 12C).

FIGS. 13A and 13B show a stellated feature on the interior of reagenttube, in cross-sectional (FIG. 13A) and plan (FIG. 13B) view.

FIG. 14 shows a sequence of pipetting operations in conjunction with areagent tube having a stellated feature.

FIG. 15 shows embodiments of a laminated layer.

FIG. 16 shows a sequence of pipetting operations in conjunction with alaminated layer.

FIGS. 17A-17D show an exemplary kit containing holders and reagents.

FIG. 18 shows a liquid dispense head.

FIGS. 19A-19C show a liquid dispense head.

FIG. 20 shows an exemplary distribution manifold.

FIG. 21 shows a scanning read-head attached to a liquid dispense head.

FIG. 22 shows a barcode scanner in cross-sectional view.

FIG. 23 shows a barcode reader positioned above a microfluidiccartridge.

FIG. 24 shows pipette tip sensors.

FIGS. 25A and 25B show an exemplary device for stripping pipette tip.

FIG. 26 shows a heater unit in perspective and cross-sectional view.

FIG. 27 shows an integrated heater and separator unit in cross-sectionalview.

FIG. 28 shows a cartridge auto-loader.

FIG. 29 shows a cartridge stacker.

FIG. 30 shows a cartridge stacker in position to deliver a cartridge toan auto-loader.

FIG. 31 shows a cartridge loading system.

FIG. 32 shows a disposal unit for used cartridges.

FIG. 33 shows a cartridge stacker in full and empty configurations.

FIG. 34 shows a microfluidic cartridge, a read-head, and a cartridgetray.

FIG. 35 shows a cross-section of a pipetting head and a cartridge inposition in a microfluidic apparatus.

FIG. 36 shows an exemplary microfluidic cartridge having a 3-layerconstruction.

FIG. 37 shows a plan of microfluidic circuitry and inlets in anexemplary multi-lane cartridge.

FIG. 38A shows an exemplary multi-lane cartridge.

FIG. 38B shows a portion of an exemplary multi-lane cartridge.

FIGS. 39A, 39B show an exemplary microfluidic network in a lane of amulti-lane cartridge;

FIGS. 40A-40C show diagrams of exemplary microfluidic valves. FIG. 40Aadditionally shows the valve in an open state, and the valve in a closedstate.

FIG. 41 shows a vent.

FIG. 42 shows an exemplary highly-multiplexed microfluidic cartridge;

FIGS. 43-46 show various aspects of exemplary highly multiplexedmicrofluidic cartridges; and

FIGS. 47A-C show various aspects of a radially configured highlymultiplexed microfluidic cartridge.

FIG. 48 shows a view in cross-section of a microfluidic cartridge.

FIGS. 49A, 49B show a PCR reaction chamber and associated heaters.

FIG. 50 shows thermal images of heater circuitry in operation.

FIGS. 51A-SIC shows various cut-away sections that can be used toimprove cooling rates during PCR thermal cycling.

FIG. 52 shows a plot of temperature against time during a PCR process,as performed on a microfluidic cartridge as described herein.

FIG. 53 shows an assembly process for a cartridge as further describedherein.

FIGS. 54A and 54B show exemplary apparatus for carrying out waxdeposition.

FIGS. 55A and 55B show exemplary deposition of wax droplets intomicrofluidic valves.

FIG. 56 shows an overlay of an array of heater elements on an exemplarymulti-lane microfluidic cartridge, wherein various microfluidic networksare visible.

FIG. 57 shows a cross-sectional view of an exemplary detector.

FIG. 58 shows a perspective view of a detector in a read-head.

FIG. 59 shows a cutaway view of an exemplary detector in a read-head.

FIG. 60 shows an exterior view of an exemplary multiplexed read-headwith an array of detectors therein.

FIG. 61 shows an cutaway view of an exemplary multiplexed read-head withan array of detectors therein.

FIG. 62 shows a block diagram of exemplary electronic circuitry inconjunction with a detector as described herein.

FIG. 63 shows an exemplary liquid dispensing system.

FIG. 64 shows an exemplary heater/separator.

FIGS. 65A and 65B show exemplary aspects of a computer-based userinterface.

FIG. 66 shows schematically layout of components of a preparatoryapparatus.

FIG. 67 shows layout of components of an exemplary preparatoryapparatus.

FIG. 68 shows schematically layout of components of a diagnosticapparatus.

FIG. 69 shows layout of components of an exemplary diagnostic apparatus.

FIGS. 70 and 71 show exterior and interior of an exemplary diagnosticapparatus.

FIGS. 72A and 72B show a thermocycling unit configured to accept amicrofluidic cartridge.

FIG. 73 shows schematically a layout of components of a high-efficiencydiagnostic apparatus.

FIG. 74 shows layout of components of an exemplary high-efficiencydiagnostic apparatus.

FIG. 75 shows a plan view of a 24-lane microfluidic cartridge.

FIG. 76 shows a perspective view of the cartridge of FIG. 75.

FIG. 77 shows an exploded view of the cartridge of FIG. 75.

FIG. 78 shows an exemplary detection unit.

FIGS. 79A, 79B show cutaway portions of the detection unit of FIG. 78.

FIGS. 80, and 81 show alignment of the detection unit with amicrofluidic cartridge.

FIGS. 82 and 83 show exterior and cutaways, respectively, of an opticsblock.

FIG. 84 shows a Scorpion reaction, schematically.

FIGS. 85A-85C show, schematically, pipette head usage during variouspreparatory processes.

FIGS. 86-91 show exemplary layouts of electronics control circuitry.

DETAILED DESCRIPTION

Nucleic acid testing (NAT) as used herein is a general term thatencompasses both DNA (Deoxyribonucleic acid) and RNA (Ribonucleic acid)testing. Exemplary protocols that are specific to RNA and to DNA aredescribed herein. It is to be understood that generalized descriptionswhere not specific to RNA or to DNA either apply to each equally or canbe readily adapted to either with minor variations of the descriptionherein as amenable to one of ordinary skill in the an. It is also to beunderstood that the terms nucleic acid and polynucleotide may be usedinterchangeably herein.

The apparatuses as described herein therefore find application toanalyzing any nucleic acid containing sample for any purpose, includingbut not limited to genetic testing, and clinical testing for variousinfectious diseases in humans. Targets for which clinical assayscurrently exist, and that may be tested for using the apparatus andmethods herein may be bacterial or viral, and include, but are notlimited to: Chlamydia Trachomatis (CT); Neisseria Gonorrhea (GC); GroupB Streptococcus; HSV; HSV Typing; CMV; Influenza A & B; MRSA; RSV; TB;Trichomonas; Adenovirus; Bordatella; BK; JC; HHV6; EBV; Enterovirus; andM. pneumonia.

The apparatus herein can be configured to run on a laboratory benchtop,or similar environment, and can test approximately 45 samples per hourwhen run continuously throughout a normal working day. This number canbe increased, according to the number of tests that can be accommodatedin a single batch, as will become clear from the description herein.Results from individual raw samples are typically available in less than1 hour.

Where used herein, the term “module” should be taken to mean an assemblyof components, each of which may have separate, distinct and/orindependent functions, but which are configured to operate together toproduce a desired result or results. It is not required that everycomponent within a module be directly connected or in directcommunication with every other. Furthermore, connectivity amongst thevarious components may be achieved with the aid of a component, such asa processor, that is external to the module.

Apparatus Overview

An apparatus having various components as further described herein canbe configured into at least two formats, preparatory and diagnostic, asshown respectively in FIGS. 1A and 11B. A schematic overview of apreparatory apparatus 981 for carrying out sample preparation as furtherdescribed herein is shown in FIG. 1A. An overview of a diagnosticapparatus 971 is shown in FIG. 1B. The geometric arrangement of thecomponents of systems 971, 981 shown in FIGS. 1A and B is exemplary andnot intended to be limiting.

A processor 980, such as a microprocessor, is configured to controlfunctions of various components of the system as shown, and is therebyin communication with each such component requiring control. It is to beunderstood that many such control functions can optionally be carriedout manually, and not under control of the processor. Furthermore, theorder in which the various functions are described, in the following, isnot limiting upon the order in which the processor executes instructionswhen the apparatus is operating. Thus, processor 980 can be configuredto receive data about a sample to be analyzed, e.g., from a samplereader 990, which may be a barcode reader, an optical character reader,or an RFID scanner (radio frequency tag reader). It is also to beunderstood that, although a single processor 980 is shown as controllingall operations of apparatus 971 and 981, such operations may bedistributed, as convenient, over more than one processor.

Processor 980 can be configured to accept user instructions from aninput 984, where such instructions may include instructions to startanalyzing the sample, and choices of operating conditions. Although notshown in FIGS. 1A and 1B, in various embodiments, input 984 can includeone or more input devices selected from the group consisting of: akeyboard, a touch-sensitive surface, a microphone, a track-pad, aretinal scanner, a holographic projection of an input device, and amouse. A suitable input device may further comprise a reader offormatted electronic media, such as, but not limited to, a flash memorycard, memory stick, USB-stick, CD, or floppy diskette. An input devicemay further comprise a security feature such as a fingerprint reader,retinal scanner, magnetic strip reader, or bar-code reader, for ensuringthat a user of the system is in fact authorized to do so, according topre-loaded identifying characteristics of authorized users. An inputdevice may additionally—and simultaneously—function as an output devicefor writing data in connection with sample analysis. For example, if aninput device is a reader of formatted electronic media, it may also be awriter of such media. Data that may be written to such media by such adevice includes, but is not limited to, environmental information, suchas temperature or humidity, pertaining to an analysis, as well as adiagnostic result, and identifying data for the sample in question.

Processor 980 can be also configured to communicate with a display 982,so that, for example, information about an analysis is transmitted tothe display and thereby communicated to a user of the system. Suchinformation includes but is not limited to: the current status of theapparatus; progress of PCR thermocycling; and a warning message in caseof malfunction of either system or cartridge. Additionally, processor980 may transmit one or more questions to be displayed on display 982that prompt a user to provide input in response thereto. Thus, incertain embodiments, input 984 and display 982 are integrated with oneanother.

Processor 980 can be optionally further configured to transmit resultsof an analysis to an output device such as a printer, a visual display,a display that utilizes a holographic projection, or a speaker, or acombination thereof.

Processor 980 can be still further optionally connected via acommunication interface such as a network interface to a computernetwork 988. The communication interface can be one or more interfacesselected from the group consisting of: a serial connection, a parallelconnection, a wireless network connection, a USB connection, and a wirednetwork connection. Thereby, when the system is suitably addressed onthe network, a remote user may access the processor and transmitinstructions, input data, or retrieve data, such as may be stored in amemory (not shown) associated with the processor, or on some othercomputer-readable medium that is in communication with the processor.The interface may also thereby permit extraction of data to a remotelocation, such as a personal computer, personal digital assistant, ornetwork storage device such as computer server or disk form. Theapparatus may further be configured to permit a user to e-mail resultsof an analysis directly to some other party, such as a healthcareprovider, or a diagnostic facility, or a patient.

Additionally, in various embodiments, the apparatus can further comprisea data storage medium configured to receive data from one or more of theprocessor, an input device, and a communication interface, the datastorage medium being one or more media selected from the groupconsisting of: a hard disk drive, an optical disk drive, a flash card,and a CD-Rom.

Processor 980 can be further configured to control various aspects ofsample preparation and diagnosis, as follows in overview, and as furtherdescribed in detail herein. In FIGS. 1A and 16B, the apparatus 981 (or971) is configured to operate in conjunction with a complementary rack970. The rack is itself configured, as further described herein, toreceive a number of biological samples 996 in a form suitable forwork-up and diagnostic analysis, and a number of holders 972 that areequipped with various reagents, pipette tips and receptacles. The rackis configured so that, during sample work-up, samples are processed inthe respective holders, the processing including being subjected,individually, to heating and cooling via heater assembly 977. Theheating functions of the heater assembly can be controlled by theprocessor 980. Heater assembly 977 operates in conjunction with aseparator 978, such as a magnetic separator, that also can be controlledby processor 980 to move into and out of close proximity to one or moreprocessing chambers associated with the holders 972, wherein particlessuch as magnetic particles are present.

Liquid dispenser 976, which similarly can be controlled by processor980, is configured to carry out various suck and dispense operations onrespective sample, fluids and reagents in the holders 972, to achieveextraction of nucleic acid from the samples. Liquid dispenser 976 cancarry out such operations on multiple holders simultaneously. Samplereader 990 is configured to transmit identifying indicia about thesample, and in some instances the holder, to processor 980. In someembodiments a sample reader is attached to the liquid dispenser and canthereby read indicia about a sample above which the liquid dispenser issituated. In other embodiments the sample reader is not attached to theliquid dispenser and is independently movable, under control of theprocessor. Liquid dispenser 976 is also configured to take aliquots offluid containing nucleic acid extracted from one or more samples anddirect them to storage area 974, which may be a cooler. Area 974contains, for example, a PCR tube corresponding to each sample. In otherembodiments, there is not a separate Area 974, but a cooler can beconfigured to cool the one or more holders 972 so that extracted nucleicacid is cooled and stored in situ rather than being transferred to aseparate location.

FIG. 1B shows a schematic embodiment of a diagnostic apparatus 971,having elements in common with apparatus 981 FIG. 1A but, in place of astorage area 974, having a receiving bay 992 in which a cartridge 994 isreceived. The receiving bay is in communication with a heater 998 thatitself can be controlled by processor 980 in such a way that specificregions of the cartridge are heated at specific times during analysis.Liquid dispenser 976 is thus configured to take aliquots of fluidcontaining nucleic acid extracted from one or more samples and directthem to respective inlets in cartridge 994. Cartridge 994 is configuredto amplify, such as by carrying out PCR, on the respective nucleicacids. The processor is also configured to control a detector 999 thatreceives an indication of a diagnosis from the cartridge 994. Thediagnosis can be transmitted to the output device 986 and/or the display982, as described hereinabove.

A suitable processor 980 can be designed and manufactured according to,respectively, design principles and semiconductor processing methodsknown in the art.

Embodiments of the apparatuses shown in outline in FIGS. 1A and 1B, aswith other exemplary embodiments described herein, is advantageousbecause they do not require locations within the apparatus suitablyconfigured for storage of reagents. Neither do embodiments of thesystem, or other exemplary embodiments herein, require inlet or outletports that are configured to receive reagents from, e.g., externallystored containers such as bottles, canisters, or reservoirs. Therefore,the apparatuses in FIGS. 1A and 1B are self-contained and operate inconjunction with holders 972, wherein the holders are pre-packaged withreagents, such as in locations within it dedicated to reagent storage.

The apparatuses of FIGS. 1A and 1B may be configured to carry outoperation in a single location, such as a laboratory setting, or may beportable so that they can accompany, e.g., a physician, or otherhealthcare professional, who may visit patients at different locations.The apparatuses are typically provided with a power-cord so that theycan accept AC power from a mains supply or generator. An optionaltransformer (not shown) built into each apparatus, or situatedexternally between a power socket and the system, transforms AC inputpower into a DC output for use by the apparatus. The apparatus may alsobe configured to operate by using one or more batteries and therefore isalso typically equipped with a battery recharging system, and variouswarning devices that alert a user if battery power is becoming too lowto reliably initiate or complete a diagnostic analysis.

The apparatuses of FIGS. 1A and 1B may further be configured, in otherembodiments, for multiplexed sample analysis and/or analysis of multiplebatches of samples, where, e.g., a single rack holds a single batch ofsamples. In one such configuration, instances of a system, as outlinedin FIG. 1B, accept and to process multiple microfluidic cartridges 994.Each component shown in FIGS. 1A and 1B may therefore be present as manytimes as there are batches of samples, though the various components maybe configured in a common housing.

In still another configuration, a system is configured to accept and toprocess multiple cartridges, but one or more components in FIGS. 1A and1B is common to multiple cartridges. For example, a single apparatus maybe configured with multiple cartridge receiving bays, but a commonprocessor, detector, and user interface suitably configured to permitconcurrent, consecutive, or simultaneous, control of the variouscartridges. It is further possible that such an embodiment, alsoutilizes a single sample reader, and a single output device.

In still another configuration, a system as shown in FIG. 1B isconfigured to accept a single cartridge, wherein the single cartridge isconfigured to process more than 1, for example, 2, 3, 4, 5, or 6,samples in parallel, and independently of one another. Exemplarytechnology for creating cartridges that can handle multiple samples isdescribed elsewhere, e.g., in U.S. application Ser. No. 60/859,284,incorporated herein by reference.

It is further consistent with the present technology that a cartridgecan be tagged, e.g., with a molecular bar-code indicative of the sample,to facilitate sample tracking, and to minimize risk of sample mix-up.Methods for such tagging are described elsewhere, e.g., in U.S. patentapplication Ser. No. 10/360,854, incorporated herein by reference.

Control electronics 840 implemented into apparatus 971 or 981, shownschematically in the block diagram in FIG. 2, can include one or morefunctions in various embodiments, for example, for main control 900,multiplexing 902, display control 904, detector control 906, and thelike. The main control function may serve as the hub of controlelectronics 840 in the apparatuses of FIGS. 1A and 1B, and can managecommunication and control of the various electronic functions. The maincontrol function can also support electrical and communicationsinterface 908 with a user or an output device such as a printer 920, aswell as optional diagnostic and safety functions. In conjunction withmain control function 900, multiplexer function 902 can control sensordata 914 and output current 916 to help control heater assembly 977. Thedisplay control function 904 can control output to and, if applicable,interpret input from touch screen LCD 846, which can thereby provide agraphical interface to the user in certain embodiments. The detectorfunction 906 can be implemented in control electronics 840 using typicalcontrol and processing circuitry to collect, digitize, filter, and/ortransmit the data from a detector 999 such as one or more fluorescencedetectors. Additional functions, not shown in FIG. 2, include but arenot limited to control functions for controlling elements in FIGS. 1Aand 1B such as a liquid dispense head, a separator, a cooler, and toaccept data from a sample reader.

An exemplary apparatus, having functions according to FIG. 1A or 1B, isshown in FIGS. 3A and 3B. The exemplary apparatus in FIGS. 3A and 3B hasa housing 985, and a cover 987, shown in a closed position in FIG. 3A,and in an open position in FIG. 3B to reveal interior features 995.Cover 987 optionally has a handle 989, shown as oval and raised from thesurface of the cover, but which may be other shapes such as square,rectangular, or circular, and which may be recessed in, or flush with,the surface of the cover. Cover 987 is shown as having a hinge, thoughother configurations such as a sliding cover are possible. Bumper 991serves to prevent the cover from falling too far backwards and/orprovides a point that holds cover 987 steady in an open position.Housing 985 is additionally shown as having one or more communicationsports 983, and one or more power ports 993, which may be positionedelsewhere, such as on the rear of the instrument.

The apparatus of FIGS. 1A and 1B may optionally comprise one or morestabilizing feet that cause the body of the device to be elevated abovea surface on which system 100 is disposed, thereby permittingventilation underneath system 100, and also providing a user with animproved ability to lift system 100. There may be 2, 3, 4, 5, or 6, ormore feet, depending upon the size of system 100. Such feet arepreferably made of rubber, or plastic, or metal, and in some embodimentsmay elevate the body of system 10 by from about 2 to about 10 mm above asurface on which it is situated.

FIG. 4 shows an exemplary configuration of a portion of an interior ofan exemplary apparatus, such as that shown in FIGS. 3A and 3B. In FIG. 4are shown a rack 970, containing a number of reagent holders 972 andpatient samples 996, as well as, in close proximity thereto, a receivingbay 992 having a cartridge 994, for performing PCR on polynucleotidesextracted from the samples.

Rack

The apparatus further comprises one or more racks configured to beinsertable into, and removable from, the apparatus, each of the racksbeing further configured to receive a plurality of reagent holders, andto receive a plurality of sample tubes, wherein the reagent holders arein one-to-one correspondence with the sample tubes, and wherein thereagent holders each contain sufficient reagents to extractpolynucleotides from a sample and place the polynucleotides into aPCR-ready form. Exemplary reagent holders are further describedelsewhere herein.

An apparatus may comprise 1, 2, 3, 4, or 6 racks, and each rack mayaccept 2, 4, 6, 8, 10, 12, 16, or 20 samples such as in sample tubes802, and a corresponding number of holders 804, each at least having oneor more pipette tips, and one or more containers for reagents.

A rack is typically configured to accept a number of reagent holders804, such as those further described herein, the rack being configuredto hold one or more such holders, either permitting access on alaboratory benchtop to reagents stored in the holders, or situated in adedicated region of the apparatus permitting the holders to be accessedby one or more other functions of the apparatus, such as automatedpipetting, heating of the process tubes, and magnetic separating ofaffinity beads.

Two perspective views of an exemplary rack 800, configured to accept 12sample tubes and 12 corresponding reagent holders, in 12 lanes, areshown in FIG. 5. A lane, as used herein in the context of a rack, is adedicated region of the rack designed to receive a sample tube andcorresponding reagent holder. Two perspective views of the sameexemplary rack, in conjunction with a heater unit, are shown in FIG. 6.

Various views of a second exemplary rack 800, also configured to accept12 sample tubes and 12 reagent holders, are shown in FIG. 7, and FIGS.8A-8K. Thus, the following views are shown: side plan (FIG. 8A); frontplan, showing sample tubes (FIG. 88); rear plan, showing reagent holders(FIG. 8C); rear elevation, showing reagent holders (FIG. 8D); frontelevation, showing sample tubes (FIG. 8E); top, showing insertion of areagent holder (FIGS. 8F and 8G); top showing slot for inserting areagent holder (FIG. 8H); top view showing registration of reagentholder (FIG. 8I); close up of rack in state of partial insertion/removalfrom apparatus (FIG. 8J); and rack held by handle, removed fromapparatus (FIG. 8K). A recessed area in a diagnostic or preparatoryapparatus, as further described herein, for accepting the exemplaryremovable rack of FIG. 7 is shown in FIG. 9. Other suitably configuredrecessed areas for receiving other racks differing in shape, appearance,and form, rather than function, are consistent with the descriptionherein.

The two exemplary racks shown in the figures being non-limiting, generalfeatures of racks contemplated herein are now described using the twoexemplary racks as illustrative thereof. For example, the embodimentsshown here, at least the first lane and the second lane are parallel toone another, a configuration that increases pipetting efficiency.Typically, when parallel to one another, pairs of adjacent sample lanesare separated by 24 mm at their respective midpoints. (Other distancesare possible, such as 18 mm apart, or 27 mm apart. The distance betweenthe midpoints in dependent on the pitch of the nozzles in the liquiddispensing head, as further described herein. Keeping the spacing inmultiples of 9 mm enables easy loading from the rack into a 96 wellplate (where typically wells are spaced apart by 9 mm). Typically, also,the rack is such that plurality of reagent holders in the plurality oflanes are maintained at the same height relative to one another.

The rack is configured to accept a reagent holder in such a way that thereagent holder snaps or locks reversibly into place, and remains steadywhile reagents are accessed in it, and while the rack is being carriedfrom one place to another or is being inserted into, or removed from,the apparatus. In each embodiment, each of the second locationscomprises a mechanical key configured to accept the reagent holder in asingle orientation. In FIG. 5, it is shown that the reagent holder(s)slide horizontally into vertically oriented slots, one per holder,located in the rack. In such an embodiment, the edge of a connectingmember on the holder engages with a complementary groove in the upperportion of a slot. In FIGS. 8F, 8G, and 8I, it is shown that the reagentholder(s) can engage with the rack via a mechanical key that keeps theholders steady and in place. For example, the mechanical key cancomprise a raised or recessed portion that, when engaging with acomplementary portion of the reagent holder, permits the reagent holderto snap into the second location. It can also be seen in the embodimentsshown that the reagent holder has a first end and a second end, and themechanical key comprises a first feature configured to engage with thefirst end, and a second feature configured to engage with the second endin such a way that a reagent holder cannot be inserted the wrong wayaround.

In certain embodiments the reagent holders each lock into place in therack, such as with a cam locking mechanism that is recognized as lockedaudibly and/or physically, or such as with a mechanical key. The rackcan be configured so that the holders, when positioned in it, arealigned for proper pipette tip pick-up using a liquid dispenser asfurther described herein. Furthermore, the second location or each lanecan be deep enough to accommodate one or more pipette tips, such ascontained in a pipette tip sheath.

In certain embodiments, the rack is configured to accept the samples inindividual sample tubes 802, each mounted adjacent to a correspondingholder 804, for example on one side of rack 800. The sample tubes can beaccessible to a sample identification verifier such as a bar codereader, as further described herein. In FIG. 5, a sample tube is held atits bottom by a cylindrical receiving member. In FIG. 7, it is shownthat a sample tube can be held at both its top and bottom, such as by arecessed portion 803 configured to receive a bottom of a sample tube,and an aperture 805 configured to hold an upper portion of the sampletube. The aperture can be a ring or an open loop, or a hole in a metalsheet. The recessed portion can be as in FIG. 7, wherein it is an angledsheet of metal housing having a hole large enough to accommodate asample tube.

The rack can be designed so that it can be easily removed from theapparatus and carried to and from the laboratory environment external tothe apparatus, such as a bench, and the apparatus, for example, topermit easy loading of the sample tube(s) and the reagent holder(s) intothe rack. In certain embodiments, the rack is designed to be stable on ahorizontal surface, and not easily toppled over during carriage, and, tothis end, the rack has one or more (such as 2, 3, 4, 6, 8) feet 809. Incertain embodiments, the rack has a handle 806 to ease lifting andmoving, and as shown in FIG. 5, the handle can be locked into a verticalposition, during carriage, also to reduce risk of the rack being toppledover. The handle can optionally have a soft grip 808 in its middle. Inthe embodiment of FIG. 7, the carrying handle is positioned about anaxis displaced from an axis passing through the center of gravity of therack when loaded, and is free to fall to a position flush with an uppersurface of the rack, under its own weight.

The embodiment of FIG. 5 has a metallic base member 810 having 4 feet811 that also serve as position locators when inserting the rack intothe dedicated portion of the apparatus. The handle is attached to thebase member. The portion of the rack 812 that accepts the samples andholders can be made of plastic, and comprises 12 slots, and may bedisposable.

In the embodiment of FIG. 7, the rack comprises a housing, a pluralityof lanes in the housing, and wherein each lane of the plurality of lanescomprises: a first location configured to accept a sample tube; and asecond location, configured to accept a reagent holder; and aregistration member complementary to a receiving bay of a diagnosticapparatus. Typically, the housing is made of a metal, such as aluminum,that is both light but also can be machined to high tolerance and issturdy enough to ensure that the rack remains stable when located in thediagnostic apparatus. The registration member in FIG. 7 comprises four(4) tight tolerance pegs 815, located one per corner of the rack. Suchpegs are such that they fit snugly and tightly into complementary holesin the receiving bay of the apparatus and thereby stabilize the rack.Other embodiments having, for example, 2, or 3, or greater than 4 suchpegs are consistent with the embodiments herein.

In particular, the housing in the embodiment of FIG. 7 comprises ahorizontal member 821, and two or more vertical members 822 connected tothe horizontal member, and is such that the second location of eachrespective lane is a recessed portion within the horizontal member. Thetwo or more vertical members 809 in the embodiment of FIG. 7 areconfigured to permit the rack to free stand thereon. The housing mayfurther comprise two or more feet or runners, attached symmetrically tothe first and second vertical members and giving the rack additionalstability when positioned on a laboratory bench top.

Furthermore, in the embodiment of FIG. 7, the housing further comprisesa plurality of spacer members 825, each of which is disposed between apair of adjacent lanes. Optionally, such spacer members may be disposedvertically between the lanes.

Although not shown in the FIGs., a rack can further comprise a laneidentifier associated with each lane. A lane identifier may be apermanent or temporary marking such as a unique number or letter, or canbe an RFID, or bar-code, or may be a colored tag unique to a particularlane.

A rack is configured so that it can be easily placed at the appropriatelocation in the instrument and gives the user positive feedback, such asaudibly or physically, that it is placed correctly. In certainembodiments, the rack can be locked into position. It is desirable thatthe rack be positioned correctly, and not permitted to move thereafter,so that movement of the liquid dispenser will not be compromised duringliquid handling operations. The rack therefore has a registration memberto ensure proper positioning. In the embodiment of FIG. 7, theregistration member comprises two or more positioning pins configured toensure that the rack can only be placed in the diagnostic apparatus in asingle orientation; and provide stability for the rack when placed inthe diagnostic apparatus. The embodiment of FIG. 7 has, optionally, asensor actuator 817 configured to indicate proper placement of the rackin the diagnostic apparatus. Such a sensor may communicate with aprocessor 980 to provide the user with a warning, such as an audiblewarning, or a visual warning communicated via an interface, if the rackis not seated correctly. It may also be configured to prevent a samplepreparation process from initiating or continuing if a seating error isdetected.

In certain embodiments, the interior of the rack around the location ofprocess tubes in the various holders is configured to have clearance fora heater assembly and/or a magnetic separator as further describedherein. For example, the rack is configured so that process chambers onthe individual holder are accepted by heater units in a heater assemblyas further described herein.

Having a removable rack enables a user to keep a next rack loaded withsamples and in line while a previous rack of samples is being preparedby the apparatus, so that the apparatus usage time is maximized.

The rack can also be conveniently cleaned outside of the instrument incase of any sample spills over it or just as a routine maintenance oflaboratory wares.

In certain embodiments the racks have one or more disposable parts.

Holder

FIGS. 10A and 10B show views of an exemplary holder 501 as furtherdescribed herein. FIG. 11 shows a plan view of another exemplary holder502, as further described herein. FIG. 12A shows an exemplary holder 503in perspective view, and FIG. 12B shows the same holder incross-sectional view. FIG. 12C shows an exploded view of the same holderas in FIGS. 12A and 12B. All of these exemplary holders, as well asothers consistent with the written description herein though not shownas specific embodiments, are now described.

The exemplary holders shown in FIGS. 10A, 10B, 11, 12A, 128, and 12C caneach be referred to as a “unitized disposable strip”, or a “unitizedstrip”, because they are intended to be used as a single unit that isconfigured to hold all of the reagents and receptacles necessary toperform a sample preparation, and because they are laid out in a stripformat. It is consistent with the description herein, though, that othergeometric arrangements of the various receptacles are contemplated, sothat the description is not limited to a linear, or strip, arrangement,but can include a circular or grid arrangement.

Some of the reagents contained in the holder are provided as liquids,and others may be provided as solids. In some embodiments, a differenttype of container or tube is used to store liquids from those that storethe solids.

The holder can be disposable, such as intended for a single use,following which it is discarded.

The holder is typically made of a plastic such as polypropylene. Theplastic is such that it has some flexibility to facilitate placementinto a rack, as further described herein. The plastic is typicallyrigid, however, so that the holder will not significantly sag or flexunder its own weight and will not easily deform during routine handlingand transport, and thus will not permit reagents to leak out from it.

The holder comprises a connecting member 10 having one or morecharacteristics as follows. Connecting member 510 serves to connectvarious components of the holder together. Connecting member 510 has anupper side 512 and, opposed to the upper side, an underside 514. In FIG.10B, a view of underside 514 is shown, having various struts 597connecting a rim of the connecting member with variously the sockets,process tube, and reagent tubes. Struts 597 are optional, and may beomitted all or in part, or may be substituted by, in all or in part,other pieces that keep the holder together.

The holder is configured to comprise: a process tube 520 affixed to theconnecting member and having an aperture 522 located in the connectingmember; at least one socket 530, located in the connecting member, thesocket configured to accept a disposable pipette tip 580; two or morereagent tubes 540 disposed on the underside of the connecting member,each of the reagent tubes having an inlet aperture 542 located in theconnecting member; and one or more receptacles 550, located in theconnecting member, wherein the one or more receptacles are eachconfigured to receive a complementary container such as a reagent tube(not shown) inserted from the upper side 512 of the connecting member.

The holder is typically such that the connecting member, process tube,and the two or more reagent tubes are made from a single piece, such asa piece of polypropylene.

The holder is also typically such that at least the process tube, andthe two or more reagent tubes are translucent.

The one or more receptacles 550 are configured to accept reagent tubesthat contain, respectively, sufficient quantities of one or morereagents typically in solid form, such as in lyophilized form, forcarrying out extraction of nucleic acid from a sample that is associatedwith the holder. The receptacles can be all of the same size and shape,or may be of different sizes and shapes from one another. Receptacles550 are shown as having open bottoms, but are not limited to suchtopologies, and may be closed other than the inlet 552 in the upper sideof connecting member 510. Preferably the receptacles 550 are configuredto accept commonly used containers in the field of laboratory analysis,or containers suitably configured for use with the holder herein. Thecontainers are typically stored separately from the holders tofacilitate sample handling, since solid reagents normally requiredifferent storage conditions from liquid reagents. In particular manysolid reagents may be extremely moisture sensitive.

The snapped-in reagent tubes containing different reagents may be ofdifferent colors, or color-coded for easy identification by the user.For example they may be made of different color material, such as tintedplastic, or may have some kind of identifying tag on them, such as acolor stripe or dot. They may also have a label printed on the side,and/or may have an identifier such as a barcode on the sealing layer onthe top.

The containers 554 received by the receptacles 550 may alternatively bean integrated part of the holder and may be the same type of containeras the waste chamber and/or the reagent tube(s), or may be differenttherefrom.

In one embodiment, the containers 554 containing lyophilized reagents,disposed in the receptacles 550 (shown, e.g., in FIGS. 12A and 12C), are0.3 ml tubes that have been further configured to have a star pattern(see FIGS. 13A and 13B) on their respective bottom interior surfaces.This is so that when a fluid has been added to the lyophilized reagents(which are dry in the initial package), a pipette tip can be bottomedout in the tube and still be able to withdraw almost the entire fluidfrom the tube, as shown in FIG. 14, during the process of nucleic acidextraction. The design of the star-pattern is further describedelsewhere herein.

The reagent tubes, such as containing the lyophilized reagents, can besealed across their tops by a metal foil, such as an aluminum foil, withno plastic lining layer, as further described herein.

The embodiments 501, 502, and 503 are shown configured with a wastechamber 560, having an inlet aperture 562 in the upper side of theconnecting member. Waste chamber 560 is optional and, in embodimentswhere it is present, is configured to receive spent liquid reagents. Inother embodiments, where it is not present, spent liquid reagents can betransferred to and disposed of at a location outside of the holder, suchas, for example, a sample tube that contained the original sample whosecontents are being analyzed. Waste chamber 560 is shown as part of anassembly comprising additionally two or more reagent tubes 540. It wouldbe understood that such an arrangement is done for convenience, e.g., ofmanufacture; other locations of the waste chamber are possible, as areembodiments in which the waste chamber is adjacent a reagent tube, butnot connected to it other than via the connecting member.

The holder is typically such that the connecting member, process tube,the two or more reagent tubes, and the waste chamber (if present) aremade from a single piece, made from a material such as polypropylene.

The embodiments 501 and 503 are shown having a pipette sheath 570. Thisis an optional component of the holders described herein. It may bepermanently or removably affixed to connecting member 510, or may beformed, e.g., moulded, as a part of a single piece assembly for theholder. For example, exploded view of holder 503 in FIG. 12C showslug-like attachments 574 on the upper surface of a removable pipettesheath 570 that engage with complementary recessed portions or holes inthe underside 514 of connecting member 510. Other configurations ofattachment are possible. Pipette sheath 570 is typically configured tosurround the at least one socket and a tip and lower portion of apipette tip when the pipette tip is stationed in the at least onesocket. In some embodiments, the at least one socket comprises foursockets. In some embodiments the at least one socket comprises two,three, five, or six sockets.

Pipette sheath 570 typically is configured to have a bottom 576 and awalled portion 578 disposed between the bottom and the connectingmember. Pipette sheath 570 may additionally and optionally have one ormore cut-out portions 572 in the wall 578, or in the bottom 576. Suchcutouts provide ventilation for the pipette tips and also reduce thetotal amount of material used in manufacture of the holder. Embodiment503 has a pipette sheath with no such cutouts. In embodiment 501, such acutout is shown as an isosceles triangle in the upper portion of thesheath; a similar shaped cutout may be round at a corresponding positionin the opposite side of the sheath, obscured from view in FIG. 10A.Other cutouts could have other triangular forms, circular, oval, square,rectangular, or other polygonal or irregular shapes, and be several,such as many, in number. The wall 578 of pipette sheath 570 may alsohave a mesh or frame like structure having fenestrations or interstices.In embodiments having a pipette sheath, a purpose of the sheath is tocatch drips from used pipette tips, and thereby to prevent cross-samplecontamination, from use of one holder to another in a similar location,and/or to any supporting rack in which the holder is situated.Typically, then, the bottom 576 is solid and bowl-shaped (concave) sothat drips are retained within it. An embodiment such as 502, having nopipette sheath, could utilize, e.g., a drip tray or a drainage outlet,suitably placed beneath pipette tips located in the one or more sockets,for the same purpose. In addition to catching drips, the pipette tipsheath prevents or inhibits the tips of other reagent holders—such asthose that are situated adjacent to the one in question in a rack asfurther described herein—from touching each other when the tips arepicked up and/or dropped off before or after some liquid processingstep. Contact between tips in adjacent holders is generally not intendedby, for example, an automated dispensing head that controls sampleprocessing on holders in parallel, but the pipette tips being long caneasily touch a tip in a nearby strip if the angle when dropping off ofthe tip deviates slightly from vertical.

The holders of embodiments 501, 502, and 503, all have a connectingmember that is configured so that the at least one socket, the one ormore receptacles, and the respective apertures of the process tube, andthe two or more reagent tubes, are all arranged linearly with respect toone another (i.e., their midpoints lie on the same axis). However, theholders herein are not limited to particular configurations ofreceptacles, waste chamber, process tube, sockets, and reagent tubes.For example, a holder may be made shorter, if some apertures arestaggered with respect to one another and occupy ‘off-axis’ positions.The various receptacles, etc., also do not need to occupy the samepositions with respect to one another as is shown in FIGS. 12A and 128,wherein the process tube is disposed approximately near the middle ofthe holder, liquid reagents are stored in receptacles mounted on oneside of the process tube, and receptacles holding solid reagents aremounted on the other side of the process tube. Thus, in FIGS. 10A, 10B,and 11, the process tube is on one end of the connecting member, and thepipette sheath is at the other end, adjacent to, in an interiorposition, a waste chamber and two or more reagent tubes. Still otherdispositions are possible, such as mounting the process tube on one endof the holder, mounting the process tube adjacent the pipette tips andpipette tip sheath (as further described herein), and mounting the wastetube adjacent the process tube. It would be understood that alternativeconfigurations of the various parts of the holder give rise only tovariations of form and can be accommodated within other variations ofthe apparatus as described, including but not limited to alternativeinstruction sets for a liquid dispensing pipette head, heater assembly,and magnetic separator, as further described herein.

Process tube 520 can also be a snap-in tube, rather than being part ofan integrated piece. Process tube 520 is typically used for variousmixing and reacting processes that occur during sample preparation. Forexample, cell lysis can occur in process tube 520, as can extraction ofnucleic acids. Process tube 520 is then advantageously positioned in alocation that minimizes, overall, pipette head moving operationsinvolved with transferring liquids to process tube 520.

Reagent tubes 540 are typically configured to hold liquid reagents, oneper tube. For example, in embodiments 501, 502, and 503, three reagenttubes are shown, containing respectively wash buffer, release buffer,and neutralization buffer, each of which is used in a sample preparationprotocol.

Reagent tubes 540 that hold liquids or liquid reagents can be sealedwith a laminate structure 598. The laminate structure typically has aheat seal layer, a plastic layer such as a layer of polypropylene, and alayer of metal such as aluminum foil, wherein the heat seal layer isadjacent the one or more reagent tubes. The additional plastic film thatis used in a laminate for receptacles that contain liquid reagents istypically to prevent liquid from contacting the aluminum.

Two embodiments of a laminate structure, differing in their layerstructures, are shown in FIG. 15. In both embodiments, the heat seallayer 602, for example made of a laquer or other such polymer with a lowmelting point, is at the bottom, adjacent to the top of the holder, whenso applied. The plastic layer 604 is typically on top of the heat seallayer, and is typically made of polypropylene, having a thickness in therange 10-50 microns. The metal layer 608 is typically on top of theplastic layer and may be a layer of Al foil bonded to the plastic layerwith a layer of adhesive 606, as in the first embodiment in FIG. 15, ormay be a layer of metal that is evaporated or sputtered into placedirectly on to the plastic layer. Exemplary thicknesses for therespective layers are shown in FIG. 15, where it is to be understoodthat variations of up to a factor of 2 in thickness are consistent withthe technology herein. In particular, the aluminum foil is 0.1-15microns thick, and the polymer layer is 15-25 microns thick in oneembodiment. In another embodiment, the aluminum is 0.1-1 microns thick,and the polymer layer is 25-30 microns thick.

The laminates deployed herein make longer term storage easier becausethe holder includes the presence of scaled lyophilized reagents as wellas liquids sealed in close proximity, which is normally hard to achieve.

In one embodiment, the tops of the reagent tubes have beveled edges sothat when an aluminum foil is heat bonded to the top, the plastic meltdoes not extend beyond the rim of the tube. This is advantageousbecause, if the plastic melt reduces the inner diameter of the tube, itwill cause interference with the pipette tip during operation. In otherembodiments, a raised flat portion 599 facilitates application andremoval of laminate 598. Raised surface 599, on the upper side of theconnecting member, and surrounding the inlet apertures to the reagenttubes and, optionally, the waste chamber, is an optional feature of theholder.

The manner in which liquid is pipetted out is such that a pipette tippiercing through the foil rips through without creating a seal aroundthe pipette tip, as in FIG. 16. Such a seal around the tip duringpipetting would be disadvantageous because a certain amount of air flowis desirable for the pipetting operation. In this instance, a seal isnot created because the laminate structure causes the pierced foil tostay in the position initially adopted when it is pierced. The upperlive panels in FIG. 16 illustrate the pipetting of a reagent out from areagent tube scaled with a laminate as further described herein. At A,the pipette tip is positioned approximately centrally above the reagenttube that contains reagent 707. At B, the pipette tip is lowered,usually controllably lowered, into the reagent tube, and in so doingpierces the foil 598. The exploded view of this area shows the edge ofthe pierced laminate to be in contact with the pipette tip at the widestportion at which it penetrates the reagent tube. At C, the pipette tipis withdrawn slightly, maintaining the tip within the bulk of thereagent 707. The exploded view shows that the pierced foil has retainedthe configuration that it adopted when it was pierced and the pipettetip descended to its deepest position within the reagent tube. At D, thepipette tip sucks up reagent 707, possibly altering its height as moreand more older people undergo such tests. At E, the pipette tip isremoved entirely from the reagent tube.

The materials of the various tubes and chambers may be configured tohave at least an interior surface smoothness and surface coating toreduce binding of DNA and other macromolecules thereto. Binding of DNAis unwanted because of the reduced sensitivity that is likely to resultin subsequent detection and analysis of the DNA that is not trapped onthe surface of the holder.

The process tube also may have a low binding surface, and allowsmagnetic beads to slide up and down the inside wall easily withoutsticking to it. Moreover, it has a hydrophobic surface coating enablinglow stiction of fluid and hence low binding of nucleic acids and othermolecules.

In some embodiments, the holder comprises a registration member such asa mechanical key. Typically such a key is part of the connecting member510. A mechanical key ensures that the holder is accepted by acomplementary member in, for example, a supporting rack or a receivingbay of an apparatus that controls pipetting operations on reagents inthe holder. A mechanical key is normally a particular-shaped cut-outthat matches a corresponding cutout or protrusion in a receivingapparatus. Thus, embodiment 501 has a mechanical key 592 that comprisesa pair of rectangular-shaped cut-outs on one end of the connectingmember. This feature as shown additionally provides for a tab by which auser may gain a suitable purchase when inserting and removing the holderinto a rack or another apparatus. Embodiments 501 and 502 also have amechanical key 590 at the other end of connecting member 510. Key 590 isan angled cutout that eases insertion of the holder into a rack, as wellas ensures a good registration therein when abutting a complementaryangled cut out in a recessed area configured to receive the holder Othervariations of a mechanical key are, of course, consistent with thedescription herein: for example, curved cutouts, or various combinationsof notches or protrusions all would facilitate secure registration ofthe holder.

In some embodiments, not shown in FIG. 10A, 10B, 11, or 12A-C, theholder further comprises an identifier affixed to the connecting member.The identifier may be a label, such as a writable label, a bar-code, a2-dimensional bar-code, or an RFID tag. The identifier can be, e.g., forthe purpose of revealing quickly what combination of reagents is presentin the holder and, thus, for what type of sample preparation protocol itis intended. The identifier may also indicate the batch from which theholder was made, for quality control or record-keeping purposes. Theidentifier may also permit a user to match a particular holder with aparticular sample.

It should also be considered consistent with the description herein thata holder additionally can be configured to accept a sample, such as in asample tube. Thus, in embodiments described elsewhere herein, a rackaccepts a number of sample tubes and a number of corresponding holdersin such a manner that the sample tubes and holders can be separately andindependently loaded from one another. Nevertheless, in otherembodiments, a holder can be configured to also accept a sample, forexample in a sample tube. And thus, a complementary rack is configuredto accept a number of holders, wherein each holder has a sample as wellas reagents and other items. In such an embodiment, the holder isconfigured so that the ample is accessible to a sample identificationverifier.

Kits

The holder described herein may be provided in a sealed pouch, to reducethe chance of air and moisture coming into contact with the reagents inthe holder. Such a sealed pouch may contain one or more of the holdersdescribed herein, such as 2, 4, 6, 8, 10, 12, 16, 20, or 24 holders.

The holder may also be provided as part of a kit for carrying out samplepreparation, wherein the kit comprises a first pouch containing one ormore of the holders described herein, each of the holders configuredwith liquid reagents for, e.g., lysis, wash, and release, and a secondpouch, having an inert atmosphere inside, and one or more reagent tubescontaining lyophilized PCR reagents, as shown in FIG. 17. Such a kit mayalso be configured to provide for analysis of multiple samples, andcontain sufficient PCR reagents (or other amplification reagents, suchas for RT-PCR, transcription mediated amplification, strand displacementamplification, NASBA, helicase dependent amplification, and otherfamiliar to one of ordinary skill in the art, and others describedherein) to process such samples, and a number of individual holders suchas 2, 4, 6, 8, 10, 12, 16, 20, or 24 holders.

Reagent Tubes

As referenced elsewhere herein, the containers 554 that containlyophilized reagents are 0.3 ml tubes that have been further configuredto have a star-shaped—or stellated—pattern (see FIGS. 13A and 13B) ontheir respective bottom interior surfaces. Still other tubes for useherein, as well as for other uses not herein described, can be similarlyconfigured. Thus, for example, the benefits afforded by the star-shapedpattern also accrue to reagent tubes that contain liquid samples thatare directly pipetted out of the tubes (as well as to those tubes thatinitially hold solids that are constituted into liquid form prior topipetting). Other size tubes that would benefit from such a star-shapedpattern have sizes in the range 0.1 ml to 0.65 ml, for example.

The star-shaped pattern ensures that when a fluid is withdrawn from thetube, a pipette tip can be bottomed out in the tube and still be able towithdraw the entire, or almost the entire fluid from the tube, as shownin FIG. 14. This is important because, when working with such smallvolumes, and when target DNA can be present in very few copies, sampleloss due to imperfections of pipetting is to be minimized to everyextent possible.

The design of the star shaped pattern is important, especially whenusing for recovery of DNA/RNA present in very small numbers in theclinical sample. The stellated pattern should enable pipetting of mostof the liquid (residual volume <1 microliter) when used with a pipettebottomed out with the bottom of the tube. Additionally, the stellatedpattern should be designed to minimize surface area as well as dead-endgrooves that tend to have two undesirable effects—to trap liquid as wellas to increase undesirable retention of polynucleotides by adsorption.

FIG. 14 is now described, as follows. FIG. 14 has a number of panels,A-G, each representing, in sequence, a stage in a pipetting operation.At A, a pipette tip 2210, containing a liquid 2211 (such as a buffersolution), is positioned directly or approximately above the center ofreagent tube 2200. The tube contains a number of lyophilized pellets2212, and is sealed by a layer 2214, such as of foil. The foil may beheat-sealed on to the top of the tube. Although a laminate layer, asfurther described herein, can be placed on the reagent tube, typically alayer of aluminum foil is adequate, where the tube contents are solid,e.g., lyophilized, reagents. In some embodiments, the top of the reagenttube has chamfer edges to reduce expansion of the top rim of the tubeduring heat sealing of a foil on the top of the tube. The tube mayfurther comprise an identifiable code, such as a 1-D or a 2-D bar-codeon the top. Such a code is useful for identifying the composition of thereagents stored within, and/or a batch number for the preparationthereof, and/or an expiry date. The code may be printed on with, forexample, an inkjet or transfer printer.

Stellated pattern 2203 on the bottom interior surface of the tube 2200is shown. At B, the pipette tip is lowered, piercing seal 2214, andbrought into a position above the particles 2212. At C the liquid 2211is discharged from the pipette tip on to the particles, dissolving thesame, as shown at D. After the particles are fully dissolved, forming asolution 2218, the pipette tip is lowered to a position where it is incontact with the stellated pattern 2203. A E, the pipette tip is causedto suck up the solution 2218, and at F, the tip may optionally dischargethe solution back into the tube. Steps E and F may be repeated, asdesired, to facilitate dissolution and mixing of the lyophilizedcomponents into solution. At step G, after sucking up as much of thesolution 2218 as is practicable into the pipette tip, the pipette tip iswithdrawn from the tube. Ideally, 100% by volume of the solution 2218 isdrawn up into the pipette tip at G. In other embodiments, and dependingupon the nature of solution 2218, at least 99% by volume of the solutionis drawn up. In still other embodiments, at least 98%, at least 97%, atleast 96%, at least 95%, and at least 90% by volume of the solution isdrawn up.

The design of the stellated or star-shaped pattern can be optimized tomaximize the flow rate of liquid through the gaps in-between a bottomedout pipette, such as a p1000 pipette, and the star pattern, and isfurther described in U.S. provisional patent application Ser. No.60/959,437, filed Jul. 13, 2007, incorporated herein by reference. Itwould be understood that, although the description herein pertains topipettes and pipette tips typically used in sample preparation ofbiological samples, the principles and detailed aspects of the designare as applicable to other types of pipette and pipette tip, and may beso-adapted.

FIG. 13A shows a cross sectional perspective view of a reagent tube 2200having side wall 2201 and bottom 2202. Interior surface 2204 of thebottom is visible. A star-shaped cutout 2203 is shown in part, as threeapical grooves.

Typically the star-shaped pattern is present as a raised portion on thelower interior surface of the tube. Thus, during manufacture of areagent tube, such as by injection moulding, an outer portion of themould is a cavity defining the exterior shape of the tube. An interiorshape of the tube is formed by a mould positioned concentrically withthe outer portion mould, and having a star-shaped structure milled outof its tip. Thus, when liquid plastic is injected into the space betweenthe two portions of the mould, the star-shape is formed as a raisedportion on the bottom interior surface of the tube.

The exemplary star pattern 2203 shown in FIG. 13B in plan view resemblesa “ship's wheel” and comprises a center 2209, a circular ring 2207centered on center 2209, and 8 radial segments configured as radialgrooves 2205. Each groove meets the other grooves at center 2209, andhas a radial end, also referred to as an apex or vertex. Star pattern2203 has 8 grooves, but it would be understood that a star patternhaving fewer or a greater number of grooves, such as 3, 4, 6, 10, or 12,would be consistent with the design herein. The number of grooves of thestar should be minimum consistent with effective liquid pipetting andalso spaced apart enough not to trap the tip of any of the pipette tipsto be used in the liquid handling applications.

Center 2209 is typically positioned coincidentally with the geometriccenter of the bottom of reagent tube 2200. The tube is typicallycircular in cross-section, so identifying its center (e.g., at acrossing point of two diameters) is normally straightforward. Center2209 may be larger than shown in FIG. 13B, such as may be a circularcutout or raised portion that exceeds in diameter of the region formedby the meeting point of grooves 2205.

Ring 2207 is an optional feature of star-shaped pattern 2203. Typicallyring 2207 is centered about center 2209, and typically it also has adimension that corresponds to the lower surface of a pipette tip. Thus,when a pipette tip ‘bottoms out’ in the bottom of reagent tube 2200, thebottom of the pipette tip rests in contact with ring 2207. Ring 2207 isthus preferably a cut-our or recessed feature that can accommodate thepipette tip and assist in guiding its positioning centrally at thebottom of the tube. In other embodiments more than one, such as 2, 3, or4 concentric rings 2207 are present.

The star pattern is configured to have dimensions that give an optimalflow-rate of liquid out of the reagent tube into a suitably positionedpipette tip. The star pattern is shown in FIG. 13B as beingsignificantly smaller in diameter than the diameter of the tube at itswidest point. The star pattern may have, in various embodiments, adiameter (measured from center 2209 to apex of a groove 2205) from 5-20%of the diameter of the reagent tube, or from 10-25% of the diameter ofthe reagent tube, or from 15-30% of the diameter of the reagent tube, orfrom 20-40% of the diameter of the reagent tube, or from 25-50% of thediameter of the reagent tube, or from 30-50% the diameter of the reagenttube, or from 40-60% the diameter of the reagent tube, or from 50-75%the diameter of the reagent tube, or from 65-90% the diameter of thereagent tube.

The grooves 2205 are thus separated by ridges (occupying the space inbetween adjacent grooves). In the embodiment shown, the grooves arenarrower (occupy a smaller radial angle) than the gaps between them. Inother embodiments, the grooves may be proportionately wider than thegaps between them. In such embodiments, it may be more appropriate todescribe them as having ridges instead of grooves. In other embodiments,the grooves and ridges that separate them are of equal widths at eachradial distance from the center.

The grooves that form the apices of the star may be rounded in theirlower surfaces, such as semi-circular in cross section, but aretypically V-shaped. They may also be trapezoid in cross-section, such ashaving a wider upper portion than the bottom, which is flat, the upperportion and the bottom being connected by sloping walls.

In some embodiments, for ease of manufacture, the grooves end on thesame level in the bottom of the tube. Thus the radial ends are alldisposed on the circumference of a circle. In other embodiments, thegrooves do not all end on the same level. For example, grooves mayalternately end on different levels, and thus the ends are alternatelydisposed on the respective circumferences of two circles that occupydifferent planes in space from one another.

Grooves 2205 are shown in FIG. 13B as having equal lengths (as measuredfrom center 2209 to apex). This need not be so. In alternativeembodiments, grooves may have different lengths from one another, forexample, as alternating lengths on alternating grooves, where there arean even number of grooves. Furthermore, apices may be rounded, ratherthan pointed.

Typically the grooves taper uniformly in width and depth from center2209 to each respective apex. Still other configurations are possible,such as a groove that follows a constant width, or depth, out to aparticular radial extent, such as 30-60% of its length, and then narrowsor becomes shallower towards its apex. Alternatively, a groove may startnarrow at center 2209, widen to a widest region near its midpoint oflength, and then narrow towards its apex. Still other possibilities, notdescribed herein, are consistent with the stellated pattern.

In a 0.3 ml tube, the width of each groove 2205 at its widest point istypically around 50 microns, and the width typically tapers uniformlyfrom a widest point, closest to or at center 2209, to the apex.

In a 0.3 ml tube, the depth of a groove at the deepest point istypically around 25-50 microns and the depth typically tapers uniformlyfrom a deepest point, closest to or at center 2209, to an apex.

In a 0.3 ml tube, the radius of the star formed from the grooves,measured as the shortest distance from center 2209 to apex, is typicallyaround 0.5 mm, but may be from 0.1-1 mm, or from 0.3-2 mm.

In another embodiment, in a 0.3 ml tube, the grooves should be roundedoff and less than 100 microns deep, or less than 50 microns deep, orless than 25 microns deep.

The stellated pattern typically has a rotation axis of symmetry, theaxis disposed perpendicular to the bottom of the tube and through center2209, so that the grooves are disposed symmetrically about the rotationaxis. By this is meant that, for a grooves, a rotation of 2π/n about thecentral (rotational) axis can bring each groove into coincidence withthe groove adjacent to it.

The stellated shape shown in FIG. 13B is not limiting in that itcomprises a number of radially disposed grooves 2205, and an optionalcircular ring 2207. Other star-shaped geometries may be used, and,depending upon case of manufacture, may be preferred. For example, astar can be created simply be superimposing two or more polygons havinga common center, but offset rotationally with respect to one anotherabout the central axis. (See, for example “star polygons” described atthe Internet site mathworld.wolfram.com/StarPolygon.html) Suchalternative manners of creating star-shaped patterns are utilizableherein.

Liquid Dispenser

In various embodiments, preparation of a PCR-ready sample for use insubsequent diagnosis using the apparatus as further described herein,can include one or more of the following steps: contacting a neutralizedpolynucleotide sample with a PCR reagent mixture comprising a polymeraseenzyme and a plurality of nucleotides (in some embodiments, the PCRreagent mixture can further include a positive control plasmid and afluorogenic hybridization probe selective for at least a portion of theplasmid); in some embodiments, the PCR reagent mixture can be in theform of one or more lyophilized pellets, as stored in a receptacle on aholder, and the method can further include reconstituting the PCR pelletwith liquid to crate a PCR reagent mixture solution. Various, such asone or more, of the liquid transfer operations associated with theforegoing steps can be accomplished by an automated pipette head.

A suitable liquid dispenser for use with the apparatus herein comprisesone or more sensors; a manifold; one or more pumps in fluidcommunication with the manifold; one or more dispense heads in fluidcommunication with the manifold; and electrical connections that acceptelectrical signals from an external controller, wherein the liquiddispenser has no inlet or outlet for fluids, other than through the oneor more pumps.

A cross-sectional view of an exemplary liquid dispenser is shown in FIG.18. The liquid dispenser is configured to carry out fluid transferoperations on two or more holders simultaneously. As shown in FIG. 18,liquid dispenser 2105 can be mounted on a gantry having three degrees oftranslational freedom. Further embodiments can comprise a gantry havingfewer than three degrees of translational freedom. The manner ofmounting can be by a mechanical fastening such as one or more screws, asshown on the loft hand side of FIG. 18. A suitable gantry comprisesthree axes of belt-driven slides actuated by encoded stepper motors. Thegentry slides can be mounted on a framework of structural angle aluminumor other equivalent material, particularly a metal or metal alloy.Slides aligned in x- and y-directions (directed out of and in the planeof FIG. 18 respectively) facilitate motion of the gantry across an arrayof holders, and in a direction along a given holder, respectively.

The z-axis of the gantry can be associated with a variable force sensorwhich can be configured to control the extent of vertical motion of thehead during tip pick-up and fluid dispensing operations. Shown in FIG.18, for example, a pipette head 1803 can be mounted such that a forceacting upwardly against the head can be sensed through a relative motionbetween the head and a force sensor. For example, when pipette head 1803forces against a disposable pipette in the rack below it, an upwardforce is transmitted causing head 1803 to torque around pivot point2102, causing set screw 2104 to press against a force sensor. In turn,the force sensor is in communication with a processor or controller thatcontrols at least the vertical motion of the liquid dispenser so that,thereby, the processor or controller can send instructions to arrest thevertical motion of the liquid dispenser upon receiving an appropriatesignal from the force sensor. An exemplary force sensor suitable for useherein is available from Honeywell; its specification is shown in anappendix hereto. The force sensor mechanism shown in FIG. 18 isexemplary and one of many possible mechanisms capable of commanding thehead during up pick-up and fluid dispensing operations. For example, asan alternative to a force sensor, a stall sensor that sensesinterruption in vertical motion of the one or more dispense heads uponcontact with a sample tube or reagent holder may be used. Accordingly,as would be understood by one of ordinary skill in the art, the liquiddispenser as described herein is not limited to the specific mechanismshown in FIG. 18.

The liquid dispenser further comprises a number of individually sprungheads 1803, wherein each head is configured to accept a pipette tip fromthe one or more pipette tips in a holder. The liquid dispenser can befurther configured such that no two heads accept pipette tips from thesame holder. FIGS. 19A-C, for example, depicts four individually sprungheads 1803, but it is to be understood that the dispenser is not limitedto this number. For example, other numbers include 2, 3, 5, 6, 8, 10, or12. Furthermore, the individually sprung heads 1803 are shown arrangedin parallel to one another, but may be configured in other arrangements.

The liquid dispenser can further comprise computer-controlled pump 2100connected to distribution manifold 1802 with related computer controlledvalving. Distribution manifold 1802 can comprise a number of valves,such as solenoid valves 1801 configured to control the flow of airthrough the pipette tips: in an exemplary embodiment, there are twovalves for each pipette, and one additional valve to vent the pump.Thus, for a liquid dispenser having four pipette heads, there are ninevalves. In another embodiment there is only one valve for each pipette,and one additional valve to vent the pump. However, the distributionmanifold is not limited to comprising exactly nine solenoid valves.

The liquid dispenser is further configured to aspirate or dispense fluidin connection with analysis or preparation of solutions of two or moresamples. The liquid dispense is also configured to dispense liquid intoa microfluidic cartridge. Additionally, the liquid dispenser isconfigured to accept or dispense, in a single operation, an amount of1.0 ml of fluid or less, such as an amount of fluid in the range 10 nl-1ml.

The liquid dispenser is configured such that pump 2100 pumps air in andout of the distribution manifold. The distribution manifold comprises amicrofluidic network that distributes air evenly amongst the one or morevalves. Thus, by controlling flow of air through the manifold andvarious valves, pressure above the pipette heads can be varied so thatliquid is drawn up into or expelled from a pipette tip attached to therespective pipette heads. In this way it is not necessary to supplycompressed air via an air hose to the liquid dispenser. Neither is itnecessary to provide liquid lines to the dispense head. Furthermore, noliquid reagents or liquid samples from the holders enters any part ofthe liquid dispenser, including the manifold. This aspect reducescomplications from introducing air bubbles into samples or liquidreagents. An exemplary configuration of a distribution manifold is shownin FIG. 20.

As shown in the various figures, the entire liquid dispenser that movesup and down the z-axis is a self-contained unit having only electricalconnections to a processor or controller, and mechanical connections tothe gantry. The translational motions in three dimensions of the liquiddispenser can be controlled by a microprocessor, such as processor 980.No fluid handling lines are associated with the dispenser. This designenables simplification of assembly of the instrument, minimizescontamination of the instrument and cross-contamination of samplesbetween different instances of operation of the apparatus, increasesefficiency of pumping (minimal dead volume) and enables easy maintenanceand repair of the device. This arrangement also enables easy upgradingof features in the dispensing device, such as individual and independentpump control for each dispenser, individual pipette attachment orremoval, ability to control the pitch of the pipettes, etc.

Another aspect of the apparatus relates to a sample identificationverifier configured to check the identity of each of the number ofnucleic-acid containing samples. Such sample identification verifierscan be optical character readers, bar code readers, or radio frequencylag readers, or other suitable readers, as available to one of ordinaryskill in the art. A sample identification verifier can be mounted on thegantry, or attached to the liquid dispenser so that it moves in concertwith the liquid dispenser. Alternatively, the sample identificationverifier can be separately mounted and can move independently of theliquid dispenser. In FIGS. 21 and 22, for example, sample identificationverifier 1701 is a bar-code reader attached to the liquid dispenser. Thefield of view or barcode scanner 1701 is non-linear, enabling it todetect light reflected by mirror 2300 from the barcoded clinical sampletube 2301 in disposable rack 2302. The barcode scanner reads the barcodeon the clinical sample tube thus identifying the presence and specificsof the sample tube. Because of use or a mirror, the scanner isconfigured either to read a bar-code printed in mirror image form (thatis thus reflected into normal form), or to read a mirror image of anormal bar-code and to convert the mirror image to unreflected form viaa computer algorithm.

Sample identification verifier is configured to communicate details oflabels that it has detected or read to a processor or controller in theapparatus, thereby permitting sample identifying information to beassociated with diagnostic results and other information relating tosample preparation, and extraction and amplification of nucleic acidtherein.

In FIG. 23, the sample identification verifier is positioned to readindicia from a microfluidic cartridge.

In certain embodiments, the liquid dispenser can also comprise one ormore sensors 2001 (e.g., infra-red sensors) each of which detects thepresence of a pipette tip in a rack. In FIG. 24, for example, aninfra-red sensor 2001 can have an infra-red emitter placed opposed toit, and the presence of disposable pipette tip 2000 obstructs the lineof sight between the emitter and the detector, thus enablingdetermination of the presence or absence of the pipette tip. Thedisposal pipettes are configured perpendicular to pipettestripper-alignment plate 2003 as further described herein.

The liquid dispenser can also operate in conjunction with a motorizedplate configured to strip the pipettes and align the pipettes duringdispensing of fluid into a microfluidic cartridge, as further describedherein.

FIGS. 25A and 25B show an exemplary device for stripping pipette tipsfrom a liquid dispenser as further described herein. The pipette tipsare aligned, all at the same pitch, above respective sockets (over apipette tip sheath) in a holder. A metal plate having elongated holeslies over the sockets. The pipette tips are inserted pert way down intothe sheath through the elongated holes, and the metal plate is movedalong in such a manner that the pipette tips are clamped by theelongated portion of the holes. When the liquid dispenser is moved up,the pipette tips become detached from their respective heads. When themetal plate is subsequently moved beck to its initial position, thepipette tips remain in place in their respective sockets.

Heater Assembly & Magnetic Separator

A cross-sectional view of a heater unit of an exemplary heater assembly1401 is shown in FIG. 18 (right hand panel). The heater assemblycomprises one or more independently, controllable heater units, each ofwhich comprises a heat block. In certain embodiments there are 2, 3, 4,5, 6, 8, 10, 12, 16, 20, 24, 25, 30, 32, 36, 40, 48, or 50 heater unitsin a heater assembly. Still other numbers of heater units, such as anynumber between 6 and 100 are consistent with the description herein. Theone or more heat blocks may be fashioned from a single piece of metal orother material, or may be made separately from one another and mountedindependently of one another or connected to one another in some way.Thus, the term heater assembly connotes a collection of heater units butdoes not require the heater units or their respective heat blocks to beattached directly or indirectly to one another. The heater assembly canbe configured so that each heater unit independently heats each of theone or more process tubes 1402, for example by permitting each of theone or more heat blocks to be independently controllable, as furtherdescribed herein. In the configuration of FIG. 26, the heater assemblycomprises one or more heat blocks 1403 each of which is configured toalign with and to deliver heat to a process tube 1402. Each heat block1403 can be optionally secured and connected to the rest of theapparatus using a strip 1408 and one or more screws 1407 or otheradhesive device. This securing mechanism is not limited to such aconfiguration.

Although a cross-sectional view of one heat block 1403 is shown in FIG.26, it should be understood that this is consistent with having multipleheat blocks aligned in parallel to one another and such that theirgeometric midpoints all lie on a single linear axis, though it is not solimited in configuration. Thus, the one or more heat blocks may bepositioned at different heights from one another, in groups or,alternately, individually, or may be staggered with respect to oneanother from left to right in FIG. 26 (right hand panel), in groups oralternately, or individually. Additionally, and in other embodiments,the heat blocks are not aligned parallel to one another but are disposedat angles relative to one another, the angles being other than 180°.Furthermore, although the heat block shown in FIG. 26 may be one ofseveral that are identical in size, it is consistent with the technologyherein that one or more heat blocks may be configured to accept and toheat process tubes of different sizes.

The exemplary heat block 1403 in FIG. 26 (right hand panel) isconfigured to have an internal cavity that partially surrounds a lowerportion of process tube 1402. In the heat block of FIG. 26, the internalcavity surrounds the lower portion of process tube 1402 on two sides butnot the front side (facing away from magnet 1404) and not the rear side(adjacent to magnet 1404). In other embodiments, heat block 1403 isconfigured to surround the bottom of process tube 1402 on three sides,including the front side. Still other configurations of heat block 1403are possible, consistent with the goals of achieving rapid and uniformheating of the contents of process tube 1402. In certain embodiments,the heat block is shaped to conform closely to the shape of process tube1402 so as to increase the surface area of the heat block that is incontact with the process tube during heating of the process tube. Thus,although exemplary heat block 1403 is shown having a conical,curve-bottomed cavity in which a complementary process tube is seated,other embodiments of heat block 1403 have, for example, a cylindricalcavity with a flat bottom. Still other embodiments of heat block 1403may have a rectilinear internal cavity such as would accommodate acuvette.

Moreover, although heat block 1403 is shown as an L-shape in FIG. 26,which aids in the transmittal of heat from heating element 1501 and insecuring the one or more heat blocks to the rest of the apparatus, itneed not be so, as further described herein. For example, in someembodiments heating element 1501 may be positioned directly underneathprocess tube 1402.

Each heat block 1403 is configured to have a low thermal mass whilestill maintaining high structural integrity and allowing a magnet toslide past the heat blocks and the process tubes with ease. A lowthermal mass is advantageous because it allows heat to be delivered ordissipated rapidly, thus increasing the heating and cooling efficiencyof the apparatus in which the heater assembly is situated. Factors thatcontribute to a low thermal mass include the material from which a heatblock is made, and the shape that it adopts. The heat blocks 1403 cantherefore be made of such materials as aluminum, silver, gold, andcopper, and alloys thereof, but are not so limited.

In one embodiment, the heat block 1403 has a mass of ˜10 grams and isconfigured to heat up liquid samples having volumes between 1.2 ml and10 μl. Heating from room temperature to 65° C. for a 1 ml biologicalsample can be achieved in less than 3 minutes, and 10 μl of an aqueousliquid such as a release buffer up to 85° C. (from 50° C.) in less than2 minutes. The heat block 1403 can cool down to 50° C. from 85° C. inless than 3 minutes. The heat block 1403 can be configured to have atemperature uniformity of 65±4° C. for heating up 1 ml of sample and85±3° C. for heating up 10 μl of release buffer. These ranges aretypical, but the heat block can be suitably scaled to heat other volumesof liquid at rates that are slower and faster than those described. Thisaspect of the technology is one aspect that contributes to achievingrapid nucleic acid extraction of multiple samples by combination ofliquid processing steps, rapid heating for lysis. DNA capture andrelease and magnetic separation, as further described herein.

Not shown in FIG. 26, the heater assembly 1401 can also optionally becontained in an enclosure that surrounds the heat blocks 1403. Theenclosure can be configured to enable sufficient air flow around theprocess tubes and so as not to significantly inhibit rate of cooling.The enclosure can have a gap between it and the heat blocks tofacilitate cooling. The enclosure can be made of plastic, but is not solimited. The enclosure is typically configured to appear aesthetic to auser.

As shown in FIG. 26, the heater assembly 1401 can also comprise one ormore heating elements (e.g., a power resistor) 1501 each of which isconfigured to thermally interface to a heat block 1403 and dissipateheat to it. For example, in one embodiment, a power resistor candissipate up to 25 Watts of power. A power resistor is advantageousbecause it is typically a low-cost alternative to a heating element.Other off-the-shelf electronic components such as power transistors mayalso be used to both sense temperature and heat. Although the heatingelement 1501 is shown placed at the bottom of the heat block 1403, itwould be understood that other configurations are consistent with theassembly described herein: for example, the heating element 1501 mightbe placed at the top or side of each heat block 1403, or directlyunderneath process tube 1402. In other embodiments, the heating elementhas other shapes and is not rectangular in cross section but may becurved, such as spherical or ellipsoidal. Additionally, the heatingelement may be moulded or shaped so that it conforms closely orapproximately to the shape of the bottom of the process tube. Not shownin FIG. 26, the heater assembly can also comprise an interface material(e.g., Berquist q-pad, or thermal grease) between the heating element1501 and the heat block 1403 to enable good thermal contact between theelement and the heat block.

In the embodiment shown in FIG. 26, the heater assembly furthercomprises one or more temperature sensors 1502, such as resistivetemperature detectors, to sense the respective temperatures of each heatblock 1403. Although a temperature sensor 1502 is shown placed at thebottom of the heat block 1403, it would be understood that otherconfigurations are consistent with the assembly described herein: forexample, the temperature sensor might be placed at the top or side ofeach heat block 1403, or closer to the bottom of process tube 1402 butnot so close as to impede uniform heating thereof. As shown in theembodiment of FIG. 26, the heater assembly can further comprise aninterface material (e.g., Berquist q-pad) 1503 configured to enable goodthermal contact between the sensor 1502 and the heat block 1403, tothereby ensure an accurate reading.

Certain embodiments of the diagnostic or preparatory apparatus hereinhave more than one heater assembly as further described herein Forexample, a single heater assembly may be configured to independentlyheat 6 or 12 process tubes, and an apparatus may be configured with twoor four such heater assemblies.

The disclosure herein further comprises a magnetic separator, configuredto separate magnetic particles, the separator comprising: one or moremagnets affixed to a supporting member; a motorized mechanism configuredto move the supporting member in such a manner that the one or moremagnets move backwards and forwards along a fixed axis, and during atleast a portion of the motion, the one or more magnets maintain closeproximity to one or more receptacles which contain the magneticparticles in solution; and control circuitry to control the motorizedmechanism.

The disclosure herein still further includes an integrated magneticseparator and heater, comprising: a heater assembly, wherein the heaterassembly comprises a plurality of independently controllable heaterunits, each of which is configured to accept and to heat one of aplurality of process tubes; one or more magnets affixed to a supportingmember, a motorized mechanism configured to move the supporting memberin such a manner that the one or more magnets move backwards andforwards along a fixed axis, and during at least a portion of the motionthe one or more magnets maintain close proximity to one or more of theprocess tubes in the heater assembly, wherein the one or more processtubes contain magnetic particles; and control circuitry to control themotorized mechanism and to control heating of the heater units.

Typically, each of the one or more receptacles is a process tube, suchas for carrying out biological reactions. In some embodiments, closeproximity can be defined as a magnet having a face less than 2 mm awayfrom the exterior surface of a process tube without being in contactwith the tube. It can still further be defined to be less than 1 mm awaywithout being in contact with the tube, or between 1 and 2 mm away.

Typically the magnetic particles are microparticles, beads, ormicrospheres capable of binding one or more biomolecules, such aspolynucleotides. Separating the particles, while in solution, typicallycomprises collecting and concentrating, or gathering, the particles intoone location in the inside of the one or more receptacles.

An exemplary magnetic separator 1400 is shown in FIG. 27, configured tooperate in conjunction with heater assembly 1401. The magnetic separator1400 is configured to move one or more magnets relative to the one ormore process tubes 1402. While the magnet 1404 shown in FIG. 27 is shownas a rectangular block, it is not so limited in shape. Moreover, theconfiguration of FIG. 27 is consistent with either having a singlemagnet that extends across all heat blocks 1403 or having multiplemagnets operating in concert and aligned to span a subset of the heatblocks, for example, aligned collinearly on the supporting member. Themagnet 1404 can be made of neodymium (e.g., from K &J Magnetics, Inc.)and can have a magnetic strength of 5,000-15,000 Gauss (Brmax). Thepoles of the magnets 1404 can be arranged such that one pole faces theheat blocks 1403 and the other faces away from the heat blocks.

Further, in the embodiment shown in FIG. 27, the magnet 1404 is mountedon a supporting member 1505 that can be raised up and down along a fixedaxis using a motorized shaft 1405. The fixed axis can be vertical. Inthe embodiment shown in FIG. 27, a geared arrangement 1406 enables themotor 1601 to be placed perpendicular to the shaft 1405, thereby savingspace in the apparatus in which magnetic separator 1400 is situated. Inother embodiments, the motor is placed underneath shaft 1405. It wouldbe understood that other configurations are consistent with the movementof the magnet relative to the process tubes, including, but not limitedto, moving the magnet from side-to-side, or bringing the magnet downfrom above. The motor can be computer controlled to run at a particularspeed; for example at a rotational speed that leads to vertical motionof the magnet in the range 1-20 mm/s. The magnetic separator can thus beconfigured to move repetitively, e.g., up an down, from side to side, orbackwards and forwards, along the same axis several times. In someembodiments there is more than one shaft that operates under motorizedcontrol. The presence of at least a second shaft has the effect ofmaking the motion of the separator more smooth. In some embodiments, thesupporting member rides on one more guiding members to ensure that thesupporting member does not, for example, tip, twist, or yaw, or undergoother internal motions while moving (other than that of controlledmotion along the axis) and thereby reduce efficacy of the separation.

The supporting member can also be configured to move the magnets betweena first position, situated away from the one or more receptacles, and asecond position situated in close proximity to the one or morereceptacles, and is further configured to move at an amplitude about thesecond position where the amplitude is smeller than a distance betweenthe first position and the second position as measured along the shall.

Shown in FIGS. 26 and 27, the heater assembly 1401 and the magneticseparator 1400 can be controlled by electronic circuitry such as onprinted circuit board 1409. The electronic circuitry 1409 can beconfigured to cause the heater assembly 1401 to apply heat independentlyto the process tubes 1402 to minimize the cost of heating and sensing.It can also be configured to cause the magnetic separator 1400 to moverepetitively relative to the process tubes 1402. The electroniccircuitry 1409 can be integrated into a single printed circuit board(PCB). During assembly, a plastic guide piece can help maintain certainspacing between individual heat blocks 1403. This design can benefitfrom use off-the-shelf electronics to control a custom arrangement ofheat blocks 1403.

Not shown in FIGS. 26 and 27, an enclosure can cover the magneticseparator 1400 and the heater assembly 1401 for protection ofsub-assemblies below and aesthetics. The enclosure can also be designedto keep the heat blocks 1403 spaced apart from one another to ensureefficiency of heating and cooling. The magnetic separator and heaterassembly can, alternatively, be enclosed by separate enclosures. The oneor more enclosures can be made of plastic.

Advantageously, the heater assembly and magnetic separator operatetogether to permit successive heating and separation operations to beperformed on liquid materials in the one or more process tubes withouttransporting either the liquid materials or the process tubes todifferent locations to perform either heating or separation. Suchoperation is also advantageous because it means that the functions ofheating and separation which, although independent of one another, areboth utilized in sample preparation may be performed with a compact andefficient apparatus.

Cartridge Autoloader

An exemplary embodiment of a PCR amplification-detection system 2900 foruse with a microfluidic cartridge is shown in FIG. 28. The system 2900performs and automates the process of PCR on multiple nucleic-acidcontaining samples in parallel. The system 2900 comprises a depository2907 for unused microfluidic cartridges, a cartridge autoloader, areceiving bay for a microfluidic cartridge, a detector, and a waste tray2903 configured to receive used microfluidic cartridges. In oneembodiment, the cartridge autoloader comprises a cartridge pack 2901,and a cartridge pusher 2904.

The system 2900, for illustration purposes, is configured so that amicrofluidic cartridge moves in a plane and in a linear manner from thedepository to the receiving bay, to the waste bin, but it need not be soarranged. For example, the waste cartridge bin 2903 can be alignedorthogonally, or any angle thereof, to the receiving bay, such asdisposed behind it. Alternatively, each element (cartridge autoloader2901, receiving bay 2902, and waste cartridge bin 2903) can beconfigured in a step-wise manner where the cartridge pack 2901 is on thesame, higher or lower level than the microfluidic PCRamplification-detection system 2902 and the microfluidic PCRamplification-detection system 2902 is on the same, higher or lowerlevel than the waste cartridge bin 2903. Another configuration could bethat each of the three elements is not arranged linearly but at an angleto one another, although within the same plane.

FIG. 28 illustrates the cartridge pack 2901 and the waste cartridge bin2903 below the plane of the receiving bay, and a detection system 2908above the plane. This configuration is exemplary and it would beunderstood that these elements may be positioned above or below theplane in other embodiments.

FIG. 29 illustrates a depository for unused microfluidic cartridges. Thedepository can be configured to accept a number of individually stackedand individually loaded cartridges, or can be configured to accept apack of cartridges. An exemplary cartridge pack has 24 cartridges. Thedepository may consist of a cage 2910 of any material that may or maynot be transparent. For example it may be made of metal or plastic. Thecartridge pack 2901 is not limited to twenty-four cartridges 106 perpack but may contain any number from 2 to 100. For example, othernumbers such as 2, 4, 8, 10, 12, 16, 20, 30, 36, 40, 48, 50, or 64 arepossible numbers of cartridges 106 per pack. Similarly, the depositorymay be configured to accept those numbers of cartridges, whenindividually stacked. In one embodiment, as in FIG. 29, each cartridge2906, individually stacked, rests on ledges 2911 that protrude from thecage 2910. However, other configurations are possible. For example, acartridge 2906 may rest on recessed grooves made within the interiorsurfaces of cage 2910. Furthermore, the cartridge pack 2901 may not needto be placed in a cage 2910. The cartridge pack 2901 may itself includethe necessary connections to bind securely to the apparatus to load thecartridges 2906.

FIG. 30 is an illustration of an exemplary initial loading position of acartridge pack 2901 in a depository when samples are loaded in thetopmost cartridge in the pack. FIG. 30 shows the cartridge pack 2901below a plane that contains a cartridge pusher. In other embodiments,the cartridge pack 2901 may be above the plane of a cartridge pusherwhere the pusher pushes the lowest cartridge out from the holder, orpartly above and partly below in a holder 2920 where a cartridge pusherpushes a cartridge from the middle of the cartridge pack 2901. In theembodiment shown, a topmost cartridge 106 is pushed along two guiderails 2905. Alternatively, there may be more or fewer guide rails (suchas one or three) or no guide rails at all so long as a cartridge 2906can be caused to move to other required positions.

An exemplary cartridge pusher 2904 is shown in FIG. 31. The cartridgepusher 2904 pushes a cartridge 2906 along guide rails 2905, which allowsa cartridge 2906 to travel to pre-calibrated positions by the mechanismof a stepper motor 2930. However, it would be understood that themechanism of transporting the cartridge 2906 is not limited to a steppermotor 2930 and thus other mechanisms are also consistent with thecartridge pusher 2904 as described herein.

FIG. 32 shows a used cartridge 2906 that has been pushed by thecartridge pusher 2904 into the waste cartridge bin 2903 after a PCRprocess has been completed. The embodiment shows a lipped handle 2940that facilitates easy handling, such as emptying, of the bin 2903.However, it would be understood that the handle 2904 is not limited tothe style and shape shown.

An exemplary cartridge pack 2901, before and after multiple PCRprocesses are completed are shown in FIG. 33. After the cartridge pusher2904 pushes a cartridge 2906 out of the cartridge pack 2901, a spring2950 at the bottom of the cartridge pack pushes against the lowersurface of the stack of cartridges and causes the topmost cartridge tobe made available for sample injection. The spring 2950 is not limitedin number or type. Thus although a single helical or coiled spring isshown, it is consistent with the description herein that more than onehelical or coiled springs could be used, such as 2, 3, or 4, and thatalternatively a sprung metal strip, or several strips, could be used.Alternatively another mechanism for forcing the cartridges upwards couldbe deployed, such as a pneumatic, hydraulic, or inflatable pressurizedcontainer, could be utilized.

It is to be noted that microfluidic cartridges, as further describedherein, that have a raised lip along their edges to permit ease ofstacking and/or storage in a pack or an auto-loader are particularlyadvantageous because the raised lips also introduce a stiffness into thecartridges and assist in keeping the fluid inlets on one cartridge awayfrom those on another cartridge during storage and transport. The raisedregions, which need not only be lips along each edge of a cartridge,also help minimize friction between the lower surface of one cartridgeand the upper surface of another.

Cartridge Receiving Bay

The present technology relates to an apparatus and related methods foramplifying, and carrying out diagnostic analyses on, nucleotides frombiological samples. The apparatus is configured to act on a disposablemicrofluidic cartridge containing multiple sample lanes in parallel, andcomprises a reusable instrument platform that can actuate on-cartridgeoperations, can detect and analyze the products of the PCR amplificationin each of the lanes separately, in all simultaneously, or in groupssimultaneously, and, optionally, can display the results on a graphicaluser interface.

FIG. 34 shows a perspective view of an exemplary cartridge 200 thatcontains multiple sample lanes, and exemplary read head 300 thatcontains detection apparatus for reading signals from cartridge 200.Also shown in FIG. 34 is a tray 110 that, optionally, can accommodatecartridge 200 prior to insertion of the cartridge in a receiving bay.The apparatus described herein is able to carry out real-time PCR on anumber of samples in cartridge 200 simultaneously. Preferably the numberof samples is 12 samples, as illustrated with exemplary cartridge 200,though other numbers of samples such as 4, 8, 10, 16, 20, 24, 25, 30,32, 36, 40, and 48 are within the scope of the present description. Inpreferred operation of the apparatus, a PCR-ready solution containingthe sample, and, optionally, one or more analyte-specific reagents(ASR's) using other components of the apparatus, as further describedherein, prior to introduction into cartridge 200.

In some embodiments, an apparatus includes a bay configured toselectively receive a microfluidic cartridge; at least one heat sourcethermally coupled to the bay; and coupled to a processor as furtherdescribed herein, wherein the heat source is configured to heatindividual sample lanes in the cartridge, and the processor isconfigured to control application of heat to the individual samplelanes, separately, in all simultaneously, or in groups simultaneously.

In some embodiments, an apparatus further includes at least one detectorconfigured to detect a polynucleotide (nucleic acid) in a sample in oneor more of the individual sample lanes, separately or simultaneously;wherein the processor is coupled to the detector to control the detectorand to receive signals from the detector.

The bay can be a portion of the apparatus that is configured toselectively receive the microfluidic cartridge. For example, the bay andthe microfluidic cartridge can be complementary in shape so that themicrofluidic cartridge is selectively received in, e.g., a singleorientation. For example, the microfluidic cartridge can have aregistration member that fits into a complementary feature of the bay.The registration member can be, for example, a cut-out on an edge of thecartridge, such as a corner that is cut-off, or one or more notches thatare made on one or more of the sides. By selectively receiving thecartridge, the bay can help a user to place the cartridge so that theapparatus can properly operate on the cartridge. In this way, error-freealignment of cartridges can be achieved. Moreover, the cartridge can bedesigned to be slightly smaller than the receiving bay by approximately200-300 micron for easy placement and removal of the cartridge. Theapparatus can further include a sensor configured to sense whether themicrofluidic cartridge is selectively received

The bay can also be configured so that various components of theapparatus that can operate on the microfluidic cartridge (heat sources,detectors, force members, and the like) are positioned to properlyoperate on the microfluidic cartridge. For example, a contact heatsource can be positioned in the bay such that it can be thermallycoupled to a distinct location at a microfluidic cartridge that isselectively received in the receiving bay.

Alternatively, in connection with alignment of microheaters in theheater module with corresponding heat-requiring microcomponents (such asvalves, pumps, gates, reaction chambers, etc), the microheaters can bedesigned to be slightly bigger than the heat requiring microfluidiccomponents so that even though the cartridge may be off-centered fromthe heater, the individual components can still function effectively.

The detector 300 can be, for example, an optical detector, as furtherdescribed herein. For example, the detector can include a light sourcethat selectively emits light in an absorption band of a fluorescent dye,and a light detector that selectively detects light in an emission bandof the fluorescent dye, wherein the fluorescent dye corresponds to afluorescent polynucleotide probe or a fragment thereof. Alternatively,for example, the optical detector can include a bandpass-filtered diodethat selectively emits light in the absorption band of the fluorescentdye and a bendpass filtered photodiode that selectively detects light inthe emission band of the fluorescent dye; or for example, the opticaldetector can be configured to independently detect a plurality offluorescent dyes having different fluorescent emission spectra, whereineach fluorescent dye corresponds to a fluorescent polynucleotide probeor a fragment thereof; or for example, the optical detector can beconfigured to independently detect a plurality or fluorescent dyes at aplurality of different locations on a microfluidic cartridge, whereineach fluorescent dye corresponds to a fluorescent polynucleotide probeor a fragment thereof in a different sample.

The heat source can be, for example, a heat source such as a resistiveheater or network of resistive heaters, a reversible heat source such asa liquid-filled heat transfer circuit or a thermoelectric element, aradiative heat source such as a xenon lamp, and the like.

In preferred embodiments, the at least one heat source can be a contactheat source selected from a resistive heater (or network thereof), aradiator, a fluidic heat exchanger and a Peltier device. The contactheat source can be configured at the receiving bay to be thermallycoupled to one or more distinct locations or a microfluidic cartridgereceived in the bay, whereby the distinct locations are selectivelyheated. At least one additional contact heat source can be included,wherein the contact heat sources are each configured at the bay to beindependently thermally coupled to a different distinct location in amicrofluidic cartridge received in the bay, whereby the distinctlocations are independently heated. The contact heat source can beconfigured to be in direct physical contact with a distinct location ofa microfluidic cartridge received in the bay. In various embodiments,each contact source heater can be configured to heat a distinct locationhaving an average diameter in 2 dimensions from about 1 millimeter (mm)to about 15 mm (typically about 1 mm to about 10 mm), or a distinctlocation having a surface area of between about 1 mm² about 225 mm²(typically between about 1 mm² and about 100 mm², or in some embodimentsbetween about 5 mm² and about 50 mm²).

In various embodiments, at least one heat source can be a radiative heatsource configured to direct heat to a distinct location of amicrofluidic cartridge received in the receiving bay.

In various embodiments, the apparatus includes one or more force membersthat are configured to apply force to thermally couple the at least oneheat source to at least a portion of the microfluidic cartridge receivedin the bay. The one or more force members can be configured to operate amechanical member at the microfluidic cartridge. At least one forcemember can be manually operated. At least one force member can bemechanically coupled to a lid at the receiving bay, whereby operation ofthe lid operates the force member.

In various embodiments, the force applied by the one or more forcemembers can result in an average pressure at an interface between aportion of the receiving bay and a portion or the microfluidic cartridgeof about 1 psi. The application of force is important to ensureconsistent thermal contact between the heater wafer and the PCR reactorand microvalves in the microfluidic cartridge.

In various embodiments, the apparatus can further include a lid at thereceiving bay, the lid being operable to at least partially excludeambient light from the bay. The lid can be, for example, a sliding lid.The lid can include the optical detector. A major face of the lid at thebay can vary from planarity by less than about 100 micrometers, forexample, less than about 25 micrometers. The lid can be configured to beremovable from the apparatus. The lid can include a latching member thatensures that the lid is securely closed before amplification reactionsare applied to the samples in the cartridge.

FIG. 35 shows a schematic cross-sectional view of part of an apparatusas described herein, showing input of sample into a cartridge 200 via apipette tip 10 (such as a disposable pipette) attached to an automateddispensing head, and an inlet 202. Although not shown, there are as manyinlets 202 as samples to be input into cartridge 200. Inlet 202 ispreferably configured to receive a pipette or the bottom end of a PCRtube and thereby accept sample for analysis with minimum waste, and withminimum introduction of air. Cartridge 200 is disposed on top of and incontact with a heater substrate 400. Read head 300 is positioned abovecartridge 200 and a cover for optics 310 restricts the amount of ambientlight that can be detected by the read head.

In various embodiments, a system as described herein can include both amicrofluidic cartridge and the diagnostic apparatus.

Microfluidic Cartridge

One aspect of the present technology relates to a microfluidic cartridgeincluding a first, second, and third, layers that together define aplurality of microfluidic networks, each network having variouscomponents configured to carry out PCR on a sample having one or morepolynucleotides whose presence is to be determined. The cartridgeincludes one or more sample lanes in parallel, wherein each lane isindependently associated with a given sample for simultaneousprocessing, and each lane contains an independently configuredmicrofluidic network. An exemplary cartridge having such a constructionis shown in FIG. 36. Such a cartridge is simple to manufacture, andpermits PCR in a concentrated reaction volume (˜4 μl) and enables rapidthermocycling, at ˜20 seconds per cycle.

Although other layers may be found in cartridges having comparableperformance and case of manufacture, the cartridge herein includesembodiments having only three layers in their construction: a substratehaving an upper side and an opposed lower side, wherein the substratecomprises a microfluidic network having a plurality of sample lanes; alaminate attached to the lower side to seal the components of themicrofluidic network, and provide an effective thermal transfer layerbetween a dedicated heating element and components in the microfluidicnetwork; and a label, attached to the upper side that also covers andseals holes that are used in the manufacturing process to loadmicrofluidic components such as valves. Thus, embodiments herein includemicrofluidic cartridges consisting of three layers, a substrate, alaminate, and a label, though other, additional, features other thanlayers may be consistent with such characterizations. Embodiments hereinfurther include microfluidic cartridges consisting essentially of threelayers, a substrate, a laminate, and a label, though other, additional,features other than layers may be consistent with suchcharacterizations. Furthermore, embodiments herein still further includemicrofluidic cartridges comprising three layers, a substrate, alaminate, and a label.

A microfluidic network can include, in fluidic communication, one ormore components selected from the group consisting of: gates, valvessuch as thermally actuated valves, channels, vents, and reactionchambers. Particular components of exemplary microfluidic networks arefurther described elsewhere herein. The cartridge typically processesthe sample by increasing the concentration of a polynucleotide to bedetermined.

A sample lane is a set of elements, controllable independently of thosein another sample lane, by which a sample can be accepted and analyzed,according to methods described herein. A lane comprises at least asample inlet, and a microfluidic component, as further described hereinin connection with a microfluidic cartridge. In some embodiments, eachmicrofluidic network additionally comprises an overflow reservoir tocontain extra liquid dispensed into the cartridge.

In various embodiments, a lane can include a sample inlet port, a firstthermally actuated valve, a second thermally actuated valve, a PCRreaction chamber, and channels connecting the inlet port to the PCRreaction chamber via the first valve, and channels connecting the PCRreaction chamber to an exit vent via the second valve. The sample inletvalve can be configured to accept a quantity of sample at a pressuredifferential compared to ambient pressure of between about 100 to 5000Pa. It should be noted that the lower the loading pressure, the higherthe fill time for a aliquot of reaction mix to fill the microfluidicnetwork. Applying more pressure will reduce the fill time, but if thetime for which the pressure is applied is not determined correctly, thesample could be blown out through the microfluidic cartridge (i an endhydrophobic vent is not present). Therefore the time for which thepressure is applied should to be properly determined, such as by methodsavailable to one of ordinary skill in the art, to prevent underfill oroverfill. In general, the fill time is inversely proportional to theviscosity of the solution. For example, FIG. 37 shows a microfluidiccartridge containing twelve independent sample lanes capable ofindependent (simultaneous or successive) processing of samples.

The microfluidic network in each lane is typically configured to carryout PCR on a PCR-ready sample, such as one containing nucleic acid (DNAor RNA) extracted from a raw biological sample using other aspects ofthe apparatus as further described herein. A PCR-ready sample is thustypically a mixture comprising the PCR reagent(s) and the neutralizedpolynucleotide sample, suitable for subjecting to thermal cyclingconditions that create PCR amplicons from the neutralized polynucleotidesample. For example, a PCR-ready sample can include a PCR reagentmixture comprising a polymerase enzyme, a positive control plasmid, afluorogenic hybridization probe selective for at least a portion of theplasmid and a plurality of nucleotides, and at least one probe that isselective for a polynucleotide sequence.

Typically, the microfluidic network is configured so that the timerequired for a microdroplet of sample to pass from the inlet to thesecond valve is less than 50% of the time required for the sample totravel up to the exit vent. Typically, the microfluidic network isdesigned to have an increased flow resistance downstream of the twovalves without increasing the total volume of the microfluidic networkin comparison to the amount required to fill from the first valve to theend vent of the network.

FIG. 38A shows a perspective view of a portion of an exemplarymicrofluidic cartridge 200 according to the present technology. Thecartridge may be referred to as a multi-lane PCR cartridge withdedicated pipette inlets 202. Shown in FIG. 38A are variousrepresentative components of cartridge 200. For example, sample inlet202 is configured to accept a syringe, a pipette, or a PCR tubecontaining a PCR ready sample. More than one inlet 202 is shown, whereinone inlet operates in conjunction with a single lane. Various componentsof microfluidic circuitry in each lane are also visible. For example,microvalves 204, and 206, and vents 208, are parts of microfluidiccircuitry in a given lane. Also shown is an ultrafast PCR reactor 210,which, as further described herein, is a microfluidic channel that islong enough to permit PCR to occur in a sample. Above PCR reactor 210 isa window 212 that permits optical detection, such as detection offluorescence from a fluorescent substance, such as a fluorogenichybridization probe, in PCR reactor 210 when a detector is situatedabove window 212.

A multi-lane cartridge is configured to accept a number of samples, inparticular embodiments 12 samples, wherein the samples include at leasta first sample and a second sample, wherein the first sample and thesecond sample each contain one or more polynucleotides in a formsuitable for amplification. The polynucleotides in question may be thesame as, or different from one another, in different lanes of acartridge. The multi-sample cartridge comprises at least a firstmicrofluidic network and a second microfluidic network, adjacent to oneanother, wherein each of the first microfluidic network and the secondmicrofluidic network is as elsewhere described herein, and wherein thefirst microfluidic network accepts the first sample, and wherein thesecond microfluidic network accepts the second sample.

The sample inlets of adjacent lanes are reasonably spaced apart from oneanother to prevent any contamination of one sample inlet from anothersample when a user introduces a sample into any one cartridge. In someembodiments, the sample inlets are configured so as to preventsubsequent inadvertent introduction of sample into a given lane after asample has already been introduced into that lane.

In some embodiments, the multi-sample cartridge has a size substantiallythe same as that of a 96-well plate as is customarily used in the art.Advantageously, then, the cartridge may be used with plate handlers usedelsewhere in the art. Still more preferably, however, the multi, samplecartridge is designed so that a spacing between the centroids of sampleinlets is 9 mm, which is an industry-recognized standard. This meansthat, in certain embodiments the center-to-center distance between inletholes in the cartridge that accept samples from PCR tubes, as furtherdescribed herein, is 9 mm. The inlet holes are manufacturedfrusto-conical in shape with an appropriate conical angle so thatindustry-standard pipette tips (2 μl, 20 μl, 200 μl, volumes, etc.) fitsnugly, entering from the widest point of the inlet. Thus, in certainembodiments, an inlet comprises an inverted frustoconical structure ofat least 1 mm height, and having a diameter at its widest point thataccepts entry of a pipette tip, of from 1-5 mm. The apparatus herein maybe adapted to suit other, later-arising, industry standards for pipettetips not otherwise described herein. Typically the volume of sampleaccepted via an inlet into a microfluidic network in a sample lane isfrom 1-20 μl, and may be from 3-5 μl. The inlet hole can be designed tofit a pipette tip snugly and to create a good seal around the pipettetip, within the cone of the inlet hole. However, the cone is designedsuch that the scaling is reversible because it is undesirable if theseal is so tight that the cartridge can be pulled away from its tray, orlocation in the receiving bay, when the pipette tips are lifted afterthe dispensing operations.

FIG. 37 shows a plan view of an exemplary microfluidic cartridge having12 lanes. The inlet ports have a 6 mm spacing, so that, when used inconjunction with an automated sample loader having 4 heads, spacedequidistantly at 9 mm apart, the inlets can be loaded in three batchesof 4 inlets: e.g., inlets 1, 4, 7, and 10 together, followed by 2, 5, 8,and 11, then finally 3, 6, 9, and 12, wherein the 12 inlets are numberedconsecutively from one side of the cartridge to the other.

FIG. 39A shows a plan view of a representative microfluidic circuitfound in one lane of a multi-lane cartridge such as shown in FIGS. 38Aand 38B. FIG. 398 shows another plan view (left panel) of anotherrepresentative microfluidic circuit found in one lane of a multi-lanecartridge such as shown in FIG. 36, and shows how the circuit is visiblethrough the cartridge construction (right panel). Other configurationsor microfluidic network would be consistent with the function of thecartridges and apparatus described herein. In sequence, sample isintroduced through liquid inlet 202, and optionally flows into a bubbleremoval vent channel 208 (which permits adventitious air bubblesintroduced into the sample during entry, to escape), and continues alonga channel 216. Typically, when using a robotic dispenser of liquidsample, the volume is dispensed accurately enough that formation ofbubbles is not a significant problem, and the presence of vent channel208 is not necessary.

Throughout the operation of cartridge 200 the fluid is manipulated as amicrodroplet (not shown in FIGS. 39A,B). Valves 204 and 206 are shown inFIG. 39A as double-valves, having a source of thermally responsivematerial (also referred to as a temperature responsive substance) oneither side of the channel where they are situated. However, valves 204and 206 may either or both be single valves that have a source ofthermally responsive material on only one side of the respectivechannels. Valves 204 and 206 are initially open, so that a microdropletof sample-containing fluid can be pumped into PCR reactor 210 from inlethole 202. Upon initiating of processing, the detector present on top ofthe PCR reactor checks for the presence of liquid in the PCR reactor,and then closes valves 204 and 206 to isolate the PCR reaction mix fromthe channels on either side.

The PCR reactor 210 is a microfluidic channel that is heated through aseries of cycles to carry out amplification of nucleotides in thesample, as further described herein. Typically the PCR reactor has avolume of 3-5 μl, in particular, 4 μl. The inside walls of the channelin the PCR reactor are made very smooth and polished to a shiny finish(for example, using a polish selected from SPI A1, SPI A2, SPI A3, SPIb1, or SPI B2) during manufacture. This is in order to minimize anymicroscopic air trapping in the surface of the PCR reactor, which wouldcausing bubbling during the thermocycling steps. The presence of bubblesespecially in the detection region of the PCR reactor might cause afalse reading for the PCR reaction. Furthermore, the PCR reactor 210 ismade shallow such that the temperature gradient across the depth of thechannel is minimized. The region of the cartridge 212 above PCR reactor210 permits a detector to monitor progress of the reaction and also todetect fluorescence from a probe that binds to a quantity of amplifiednucleotide. The region 212 is made of thinner material than the rest ofthe cartridge so as to permit the PCR reactor to be more responsive to aheating cycle (for example, to rapidly heat and cool betweentemperatures appropriate for denaturing and annealing steps), and so asto reduce glare, autofluorescence, and undue absorption of fluorescence.Both valves 204 and 206 are closed prior to thermocycling to prevent anyevaporation of liquid, bubble generation, or movement of fluid from thePCR reactor.

End vent 214 prevents a user from introducing any excess amount ofliquid into the microfluidic cartridge, as well as playing a role ofcontaining any sample from spilling over to unintended parts of thecartridge. A user may input sample volumes as small as an amount to fillfrom the bubble removal vent to the middle of the PCR reactor, or up tovalve 204 or beyond valve 204. The use of microvalves prevents both lossof liquid or vapor thereby enabling even a partially filled reactor tosuccessfully complete a PCR thermocycling reaction. The application ofpressure (such as ˜1 psi) to contact the cartridge to the heater of theinstrument assists in achieving better thermal contact between theheater and the heat-receivable parts of the cartridge, and also preventsthe bottom laminate structure from expanding, as would happen if the PCRchannel was partially filled with liquid and the entrapped air would bethermally expanded during thermocycling.

In various embodiments, the microfluidic network can optionally includeat least one hydrophobic vent additional to the end vent.

After PCR has been carried out on a sample, and presence or absence of apolynucleotide of interest has been determined, it is preferred that theamplified sample remains on the cartridge and that the cartridge iseither used again (if one or more lanes remain open), or disposed of.Should a user wish to run a post amplification analysis, such as gelelectrophoresis the user may pierce a hole through the laminate of thecartridge, and recover an amount—typically about 1.5 microliter—of PCRproduct. The user may also place the individual PCR lane on a specialnarrow heated plate, maintained at a temperature to melt the wax in thevalve, and then aspirate the reacted sample from the inlet hole of thatPCR lane.

In various embodiments, the microfluidic network can optionally includeat least one reservoir configured to contain waste.

In various embodiments, the microfluidic cartridge can further include alabel, such as a computer-readable or scannable label. For example, thelabel can be a bar code, a radio frequency tag, or one or morecomputer-readable, or optically scannable, characters. The label can bepositioned such that it can be read by a sample identification verifieras further described herein.

In various embodiments, during transport sad storage, the microfluidiccartridge can be further surrounded by a sealed pouch. The microfluidiccartridge can be sealed in the pouch with an inert gas. The microfluidiccartridge can be disposable.

Microfluidic cartridge 200 can be fabricated as desired. Typically, themicrofluidic cartridge layer includes a layer of polypropylene or otherplastic label with pressure sensitive adhesive (typically between about50 and 150 microns thick) configured to seal the wax loading holes ofthe valves, trap air used for valve actuation, and serve as a locationfor operator markings. This layer can be in two separate pieces, thoughit would be understood by one of ordinary skill in the an that in manyembodiments a single piece layer would be appropriate.

The microfluidic substrate layer, is typically injection molded opt of aplastic, preferably a zeonor plastic (cyclic olefin polymer), having aPCR channel and valve channels on a first side, and vent channels andvarious inlet holes, including wax loading holes and liquid inlet holes,on a second side (disposed toward the label). Typically, all of themicrofluidic networks together, including the PCR reactors, the inletholes and the valves for isolating the PCR reaction chambers, aredefined in a single substrate. The substrate is made of a material thatconfers rigidity on the substrate and cartridge, and is impervious toair or liquid, so that entry or exit of air or liquid during operationof the cartridge is only possible through the inlet or the vent.

Channels of a microfluidic network in a lane of cartridge 200 typicallyhave at least one sub-millimeter cross-sectional dimension. For example,channels of such a network may have a width and/or a depth or about 1 mmor less (e.g., about 750 microns or less, about 500 microns, or less,about 250 microns or less).

The cartridge can further include a heat sealable laminate layer 222(typically between about 100 and about 125 microns thick) attached tothe bottom surface of the microfluidic substrate using, for example,heat bonding, pressure bonding, or a combination thereof. The laminatelayer 222 may also be made from a material that has an adhesive costingon one side only, that side being the side that contacts the undersideof the microfluidic substrate. This layer may be made from a singlecoated tape having a layer of Adhesive 420, made by 3M. Exemplary tapesinclude single-sided variants of double sided tapes having product nos.9783, 9795, and 97958, and available from 3M. Other acceptable layersmay include tapes based on micro-capsule based adhesives.

In use, cartridge 200 is typically thermally associated with an array ofheat sources configured to operate the components (e.g., valves, gates,and processing region 210) of the device. In some embodiments, the heatsources are operated by an operating system, which operates the deviceduring use. The operating system includes a processor (e.g., a computer)configured to actuate the heat sources according to a desired protocol.Processors configured to operate microfluidic devices are described in,e.g., U.S. application Ser. No. 09/819,105, filed Mar. 28, 2001, whichapplication is incorporated herein by reference.

Table 1 outlines volumes, pumping pressures, and operation timesassociated with various components of a microfluidic cartridge.

TABLE 1 Operation Pumping Pressure Displacement Volume Time of OperationMixing displacements   ~2 psi 10-25 μl  1-2 minutes Moving valve wax~1-2 psi   <1 μl 5-15 seconds plugs Operation Pump Used Pump Design PumpActuation Mixing displacements Expancel Pump Same as above Same as aboveMoving valve wax Thermopneumatic 1 μl of trapped air Heat trapped air toplugs pump ~70-90 C.

In some embodiments, a microfluidic cartridge further comprises aregistration member that ensures that the cartridge is received by acomplementary diagnostic apparatus in a single orientation, for example,in a receiving bay of the apparatus. The registration member may be asimple cut-out from an edge or a corner of the cartridge (as shown inFIG. 38A), or may be a series of notches, or some other configuration ofshapes that require a unique orientation of placement in the apparatus.

In some embodiments, the microfluidic cartridge comprises two or morepositioning elements, or fiducials, for use when filling the valves withthermally responsive material. The positioning elements may be locatedon the substrate, typically the upper face thereof.

The microfluidic cartridges may also be stackable, such as for easystorage or transport, or may be configured to be received by a loadingdevice, as further described herein, that holds a plurality ofcartridges in close proximity to one another, but without being incontact. In order to accomplish either or both of these characteristics,the substrate may comprise two ridges, one of each situated along eachof two opposite edges of the cartridge, the ridges disposed on the upperside of the substrate. Thus, where a cartridge has a rectangular aspect(ignoring any registration member or mechanical key), the two ridges maybe situated along the long side, or along the short side, of thecartridge.

Valves

A valve is a microfluidic component that has a normally open stateallowing material to pass along a channel from a position on one side ofthe valve (e.g., upstream of the valve) to a position on the other sideof the valve (e.g., downstream of the valve). An exemplary double valveis shown in FIG. 40A. A double valve has two channels, one on eitherside of the channel whose flow it regulates, whereas a single valve hasjust one channel, disposed on one side of the channel whose flow itregulates.

Upon actuation, e.g., by application of heat, the valve transitions to aclosed state that prevents material, such as a microdroplet of PCR-readysample, from passing along the channel from one side of the valve to theother. For example, a valve includes one or more masses of a thermallyresponsive substance (TRS) that is relatively immobile at a firsttemperature and more mobile at a second temperature. A mass of TRS canbe an essentially solid mass or an agglomeration of smaller particlesthat cooperate to obstruct the passage upon actuation. Examples of TRS'sinclude a eutectic alloy (e.g., a solder), wax (e.g., an olefin),polymers, plastics, and combinations thereof. The first and secondtemperatures are insufficiently high to damage materials, such aspolymer layers of a microfluidic cartridge in which the valve issituated. Generally, the second temperature is less than about 90° C.and the first temperature is less than the second temperature (e.g.,about 70° C. or less).

For each mass associated with a valve, a chamber is in gaseouscommunication with the mass. Upon heating gas (e.g., air) in thechamber(s) and heating the one or more masses of TRS to the secondtemperature, gas pressure within a chamber moves the corresponding massinto the channel obstructing material from passing therealong. Othervalves of the network have the same structure and operate in the samefashion as the valves described herein.

In order to make the valve sealing very robust and reliable, the flowchannel at the valve junction is made narrow (150 μm wide and 150 μmdeep or narrower) and the constricted channel is made at least 0.5 or 1mm long such that the wax seals up a long narrow channel therebyreducing any leakage through the walls of the channel. In the case of abad seal, there is leakage of fluid around the walls of the channel,past the wax. So the flow channel is narrowed as much as possible, andmade longer, e.g., as long as ˜1 mm. The valve operates by heating airin the wax-loading port, which forces the wax forwards in a manner sothat it does not come back to its original position. In this way, bothair and wax are heated during operation of the valve.

In various embodiments, the microfluidic network can include a bentvalve as shown in FIG. 32B (as a single valve) to reduce the footprintof the valve on the cartridge and hence reduce cost per pan formanufacturing highly dense microfluidic substrates. In the valve of FIG.40B, the loading hole for TRS is in the center of the valve; thestructures at either end are an inlet and an outlet and are shown forillustrative purposes only. Single valve shown.

In various embodiments, the network can include a curved valve as shownin FIG. 40C, also as a single valve, in order to reduce the effectivecross-section of the microvalve, enabling manufacture of cheaper densemicrofluidic devices.

Vents

A hydrophobic vent (e.g., a vent in FIG. 41) is a structure that permitsgas to exit a channel while limiting (e.g., preventing) liquid fromexiting the channel. Typically, hydrophobic vents include a layer ofporous hydrophobic material (e.g., a porous filter such as a poroushydrophobic membrane from Osmonics) that defines a wall of the channel.As discussed herein, hydrophobic vents can be used to position amicrodroplet of sample at a desired location within a microfluidicnetwork.

The hydrophobic vents or the cartridge are preferably constructed sothat the amount of air that escapes through them is maximized whileminimizing the volume of the channel below the vent surface.Accordingly, it is preferable that the vent is constructed so as to havea hydrophobic membrane of large surface area and a shallow cross sectionof the microchannel below the vent surface.

Bubble removal hydrophobic vents typically have a length of at leastabout 2.5 mm (e.g., at least about 5 mm, at least about 7.5 mm) along achannel. The length of the hydrophobic vent is typically at least about5 times (e.g., at least about 10 times, at least about 20 times) largerthan a depth of the channel within the hydrophobic vent. For example, insome embodiments, the channel depth within the hydrophobic vent is about300 microns or less (e.g., about 250 microns or less, about 200 micronsor less, about 150 microns or loss). Bubble vents are optional in themicrofluidic networks of the microfluidic cartridges described herein.

The depth of the channel within the hydrophobic vent is typically about75% or less (e.g., about 65% or less, about 60% or less) of than thedepth of the channel upstream and downstream of the hydrophobic vent.For example, in some embodiments the channel depth within thehydrophobic vent is about 150 microns and the channel depth upstream anddownstream of the hydrophobic vent is about 250 microns.

A width of the channel within the hydrophobic vent is typically at leastabout 25% wider (e.g., at least about 50% wider) than a width of thechannel upstream from the vent and downstream from the vent. Forexample, in an exemplary embodiment, the width of the channel within thehydrophobic vent is about 400 microns and the width of the channelupstream and downstream from the vent is about 250 microns.

Highly Multiplexed Embodiment

Embodiments of the apparatus and cartridge described herein may beconstructed that have high-density microfluidic circuitry on a singlecartridge that thereby permit processing of multiple samples inparallel, or in sequence, on a single cartridge. Preferred numbers ofsuch multiple samples include 36, 40, 48, 50, 64, 72, 80, 96, and 100,but it would be understood that still other numbers are consistent withthe apparatus and cartridge herein, where doomed convenient andpractical.

Accordingly, different configurations of lanes, sample inlets, andassociated heater networks are contemplated that can facilitateprocessing such numbers of samples on a single cartridge are within thescope of the instant disclosure. Similarly, alternative configurationsor detectors for use in conjunction with such a highly multiplexedcartridge are also within the scope of the description herein.

In an exemplary embodiment, a highly multiplexed cartridge has 48 PCRchannels, and has independent control of each valve in the channel, with2 banks of thermocycling protocol per channel, as shown in FIG. 43. Inthe embodiment in FIG. 43, the heaters are arranged in three arrays.Heaters in two separate glass regions only apply heat to valves in themicrofluidic networks in each lane. Because of the low thermalconductivity of glass, the individual valves may be heated separatelyfrom one another. This permits samples to be loaded into the cartridgeat different times, and passed to the PCR reaction chambersindependently of one another. The PCR heaters are mounted on a siliconsubstrate—and are not readily heated individually, but thereby permitbatch processing of PCR samples, where multiple samples from differentlanes are amplified by the same set of heating/cooling cycles. It ispreferable for the PCR heaters to be arranged in 2 banks (the heaterarrays on the left and right are not in electrical communication withone another), thereby permitting a separate degree of sample control.

FIG. 42 shows a representative cartridge, revealing an inletconfiguration for a 48-sample cartridge. The inlet configuration iscompatible with an automatic pipetting machine that has dispensing headssituated at a 9 mm spacing. For example, such a machine having 4 headscan load 4 inlets at once, in 12 discrete steps, for the cartridge ofFIG. 42.

FIG. 44 shows, in close, up an exemplary spacing of valves and lanes inadjacent lanes of a multi-sample microfluidic cartridge.

FIGS. 45 and 46 show close-ups of, respectively, heater arrays, andinlets, of the exemplary cartridge shown in FIG. 44.

FIGS. 47A-47C show various views of an embodiment of aradially-configured highly-multiplexed cartridge, having a number ofinlets, microfluidic lanes, and PCR reaction zones.

The various embodiments shown in FIGS. 42-47C are compatible with liquiddispensers, receiving bays, and detectors that are configureddifferently from the specific examples described herein.

In another preferred embodiment (not shown in the FIGs.), a cartridgeand apparatus is configured so that the read-head does not cover thesample inlets, thereby permitting loading of separate samples whileother samples are undergoing PCR thermocycling.

Heater Configurations to Ensure Uniform Heating of a Region

Another aspect of the apparatus described herein relates to a method andapparatus for uniformly controlling the heating of a region of amicrofluidic network that includes but is not limited to one or moremicrofluidic components. In an exemplary embodiment, multiple heaterscan be configured to simultaneously and uniformly heat a region, such asthe PCR reaction zone, of the microfluidic cartridge.

In preferred embodiments, a microfluidic cartridge having a microfluidicnetwork comprising one or more microfluidic components is brought intocontact with a heat source, within a suitably configured apparatus. Theheat source is configured so that particular heating elements aresituated to heat specific components of the microfluidic network of thecartridge.

FIG. 48 shows a cross-sectional view of an exemplary microfluidiccartridge to show relative location of PCR channel in relation to theheaters when the cartridge is placed in the instrument. The view in FIG.48 is also referred to as a sectional-isometric view of the cartridgelying over the heater wafer. A window 903 above the PCR channel in thecartridge is shown in perspective view. PCR channel 901 (for example,150μ deep×700μ wide), is shown in an upper layer of the cartridge. Alaminate layer 905 of the cartridge (for example, 125μ thick) isdirectly under the PCR channel 901. A further layer of thermal interfacelaminate 907 on the cartridge (for example, 125μ thick) lies directlyunder the laminate layer 905. Heaters are situated in a further layer913 directly under the thermal interface laminate. The heaters arephotolithographically defined and etched metal layers of gold (typicallyabout 3,000 Å thick). Layers of 400 Å of TiW are deposited on top andbottom of the gold layer to serve as an adhesion layer. The substrateused is glass, fused silica or quartz wafer having a thickness of 0.4mm, 0.5 mm or 0.7 mm or 1 mm. A thin electrically-insulative layer of 2μm silicon oxide serves as an insulative layer on top of the metallayer. Additional thin electrically insulative layers such as 2-4 μm ofParylene may also be deposited on top of the Silicon oxide surface. Twolong heaters 909 and 911, as further described herein, are also shown.

Referring to FIGS. 49A and 49B, the PCR reaction zone 1001, typicallyhaving a volume ˜1.6 μl, is configured with a long side and a shortside, each with an associated heating element. The apparatus thereforepreferably includes four heaters disposed along the sides of, andconfigured to heat, the PCR reaction zone, as shown in the exemplaryembodiment of FIG. 38A: long top heater 1005, long bottom heater 1003,short left heater 1007, and short right heater 1009. The small gapbetween long top heater 1005 and long bottom heater 1003 results in anegligible temperature gradient (less than 1° C. across the width of thePCR channel at any point along the length of the PCR reaction zone) andtherefore an effectively uniform temperature throughout the PCR reactionzone. The heaters on the short edges of the PCR reactor provide heat tocounteract the gradient created by the two long heaters from the centerof the reactor to the edge of the reactor. It would be understood by oneof ordinary skill in the art that still other configurations of one ormore heater(s) situated about a PCR reaction zone are consistent withthe methods and apparatus described herein. For example, a ‘long’ sideof the reaction zone can be configured to be heated by two or moreheaters. Specific orientations and configurations of heaters are used tocreate uniform zones of heating even on substrates having poor thermalconductivity because the poor thermal conductivity of glass, or quartz,or fused silica substrates is utilized to help in the independentoperation of various microfluidic components such as valves andindependent operation of the various PCR lanes.

In preferred embodiments, each heater has an associated temperaturesensor. In the embodiment of FIG. 49A, a single temperature sensor 1011is used for both long heaters. A temperature sensor 1013 for short leftheater, and a temperature sensor 1015 for short right heater are alsoshown. The temperature sensor in the middle of the reactor is used toprovide feedback and control the amount of power supplied to the twolong heaters, whereas each of the short heaters has a dedicatedtemperature sensor placed adjacent to it in order to control it. Asfurther described herein, temperature sensors are preferably configuredto transmit information about temperature in their vicinity to theprocessor at such times as the heaters are not receiving current thatcauses them to heat. This can be achieved with appropriate control ofcurrent cycles.

In order to reduce the number of sensor or heater elements required tocontrol a PCR heater, we may use the heaters to sense as well as heat,and thereby obviate the need to have a separate dedicated sensor foreach heater. In another embodiment, each of the four heaters may bedesigned to have an appropriate wattage, and connect the four heaters inseries or in parallel to reduce the number ofelectronically-controllable elements from 4 to just 1, thereby reducingthe burden on the electronics.

FIG. 49B shows expanded views of heaters and temperature sensors used inconjunction with a PCR reaction zone of FIG. 49A. Temperature sensors1001 and 1013 are designed to have a room temperature resistance ofapproximately 200-300 ohms. This value of resistance is determined bycontrolling the thickness of the metal layer deposited (e.g., a sandwichof 400 Å TiW/3000 Å Au/400 Å TiW), and etching the winding metal line tohave a width of approximately 10-25 μm and 20-40 mm length. The use ofmetal in this layer gives it temperature coefficient of resistivity ofthe order of 0.5-20° C./ohms, preferably in the range of 1.5-3° C./ohms.Measuring the resistance at higher temperatures will enabledetermination of the exact temperature of the location of these sensors.

The configuration for uniform heating, shown in FIG. 49A for a singlePCR reaction one, can be applied to a multi-lane PCR cartridge in whichmultiple independent PCR reactions occur.

Each heater can be independently controlled by a processor and/orcontrol circuitry used in conjunction with the apparatus describedherein. FIG. 50 shows thermal images, from the top surface of amicrofluidic cartridge having heaters configured as in FIGS. 49A and49B, when each heater in turn is activated, as follows: (A): Long Toponly; (B) Long Bottom only; (C) Short Left only; (D) Short Right only;and (E) All Four Heaters on. Panel (F) shows a view of the reaction zoneand haters on the same scale as the other image panels in FIG. 50. Alsoshown in the figure is a temperature bar.

Use of Cutaways in Cartridge Substrate to Improve Rate of Cooling DuringPCR Cycling

During a PCR amplification of a nucleotide sample, a number of thermalcycles are carried out. For improved efficiency, the cooling betweeneach application of heat is preferably as rapid as possible. Improvedrate of cooling can be achieved with various modifications to theheating substrate, as shown in FIGS. 51A-51C.

One way to achieve rapid cooling is to cutaway portions of themicrofluidic cartridge substrate, as shown in FIG. 51A. The upper panelof FIG. 8 IA is a cross-section of an exemplary microfluidic cartridgetaken along the dashed line A-A′ as marked on the lower panel of FIG.51A. PCR reaction zone 901, and representative heaters 1003 are shown.Also shown are two cutaway portions, one of which labeled 1201, that aresituated alongside the heaters that are situated along the long side ofthe PCR reaction zone. Cutaway portions such as 1201 reduce the thermalmass of the cartridge, and also permit air to circulate within thecutaway portions. Both of these aspects permit heat to be conducted awayquickly from the immediate vicinity of the PCR reaction zone. Otherconfigurations of cutouts, such as in shape, position, and number, areconsistent with the present technology.

Another way to achieve rapid cooling is to cutaway portions of theheater substrate, as shown in FIG. 51B. The lower panel of FIG. 51B is across-section of an exemplary microfluidic cartridge and heatersubstrate taken along the dashed line A-A′ as marked on the upper panelof FIG. 51B. PCR reaction zone 901, and representative heaters 1003 areshown. Also shown are four cutaway portions, one of which labeled 1205,that are situated alongside the heaters that are situated along the longside of the PCR reaction zone. Cutaway portions such as 1205 reduce thethermal mass of the heater substrate, and also permit air to circulatewithin the cutaway portions. Both of these aspects permit heat to beconducted away quickly from the immediate vicinity of the PCR reactionzone. Four separate cutaway portions are shown in FIG. 51B so thatcontrol circuitry to the various heaters is not disrupted. Otherconfigurations of cutouts, such as in shape, position, and number, areconsistent with the present technology. These cutouts may be created bya method selected from: selective etching using wet etching processes,deep reactive ion etching, selective etching using CO₂ laser orfemtosecond laser (to prevent surface cracks or stress near thesurface), selective mechanical drilling, selective ultrasonic drilling,or selective abrasive particle blasting. Care has to be taken tomaintain mechanically integrity of the heater while reducing as muchmaterial as possible.

FIG. 81C shows a combination of cutouts and use of ambient air coolingto increase the cooling rate during the cooling stage of thermocycling.A substantial amount of cooling happens by convective loss from thebottom surface of the heater surface to ambient air. The driving forcefor this convective loss is the differential in temperatures between theglass surface and the air temperature. By decreasing the ambient airtemperature by use of, for example, a peltier cooler, the rate ofcooling can be increased. The convective heat loss may also be increasedby keeping the air at a velocity higher than zero.

An example of thermal cycling performance obtained with a configurationas described herein, is shown in FIG. 52 for a protocol that is set toheat up to 92° C., and stay there for 1 second, then cool to 62° C., andstay for 10 seconds. Cycle time is about 29 seconds, with 8 secondsrequired to heat from 62° C. and stabilize at 92° C., and 10 secondsrequired to cool from 92° C., and stabilize at 62° C.

Manufacturing Process for Cartridge

FIG. 53 shows a flow-chart 2800 for an assembly process for an exemplarycartridge as further described herein. It would be understood by one ofordinary skill in the art, both that various steps may be performed in adifferent order from that set forth in FIG. 53, and additionally thatany given step may be carried out by alternative methods to those setforth in the figure. It would also be understood that, where separatesteps are illustrated for carrying out two or more functions, suchfunctions may be performed synchronously and combined into single stepsand be consistent with the overall process described herein.

At 2802, a laminate layer is applied to a microfluidic substrate thathas previously been engineered to have a microfluidic networkconstructed in it; edges are trimmed from the laminate where they spillover the bounds of the substrate.

At 2804, wax is dispensed and loaded into the microvalves of themicrofluidic network in the microfluidic substrate. An exemplary processfor carrying this out is further described herein.

At 2806, the cartridge is inspected to ensure that wax from step 2804 isloaded properly and that the laminate from step 2802 adheres properly tothe microfluidic substrate. If a substrate does not satisfy either orboth of these tests, it is discarded. If substrates repeatedly faileither or both of these tests, then the wax dispensing, or laminateapplication steps, as applicable, are reviewed.

At 2808, a hydrophobic vent membrane is applied to, and heat bonded to,the top of the microfluidic substrate over the wax valves, and on theopposite face of the substrate from the laminate. Edges of the membranethat are in excess of the boundary of the substrate are trimmed.

At 2810, the assembly is inspected to ensure that the hydrophobic ventmembrane is bonded well to the microfluidic substrate withoutheat-clogging the microfluidic channels. If any of the channels isblocked, or if the bond between the membrane and the substrate isimperfect, the assembly is discarded, and, in the case of repeateddiscard events, the foregoing process step is reviewed.

At 2812, a thermally conductive pad layer is applied to the bottomlaminate of the cartridge.

At 2814, two label strips are applied to the top of the microfluidicsubstrate, one to cover the valves, and a second to protect the ventmembranes. It would be understood that a single label strip may bedevised to fulfill both of these roles.

At 2816, additional labels are printed or applied to show identifyingcharacteristics, such as a barcode #, lot # and expiry date on thecartridge. Preferably one or more or these labels has a space and awritable surface that permits a user to make an identifying annotationon the label, by hand.

At 2818, to facilitate transport and delivery to a customer, assembledand labeled cartridges are stacked and pack cartridges in groups, suchas groups of 25, or groups of 10, or groups of 20, or groups of 50.Preferably the packaging is via an inert and/or moisture-free medium.

Exemplary Wax-Deposition Process

Deposition of wax in valves of the microfluidic network, as at step 2804may be carried out with the exemplary equipment shown in FIGS. 54A and54B. The DispenseJct Series DJ-9000 (FIGS. 54A and 548) is a non-contactdispenser that provides high-speed delivery and exceptional volumetriccontrol for various fluids, including surface mount adhesive, underfill,encapsulants, conformal coating, UV adhesives, and silver epoxy. TheDJ-9000 jets in tight spaces as small as 200 micrometers and createsfillet wet-out widths as small as 300 micrometers on the dispensed sideof a substrate such as a die. It dispenses fluid either as discrete dotsor a rapid succession of dots to form a 100-micron (4 mil) diameterstream of fluid from the nozzle. It is fully compatible with othercommercially available systems such as the Asymtek Century C-718/C-720,Millennium M-2000, and Axiom X-1000 Series Dispensing Systems.

A DJ-9000 is manufactured by Asymtek under manufacturing quality controlstandards aim to provide precise and reliable performance.Representative specifications of the apparatus are as follows.

Characteristic Specification Size Width: 35 mm Height: 110 mm Depth: 100mm Weight 400 grams - dry Feed Tube Assembly Nylon - FittingPolyurethane - Tube Fluid Chamber Type 303 Stainless Steel Seat andNozzle 300/400 Series S/S, Carbide Needle Assembly 52100 Bearing Steel -Shaft Hard Chrome Plate Carbide - Tip Fluid Seal PEEK/Stainless SteelFluid Chamber 0-Ring Ethylene Propylene Jet Body 6061-T6 Aluminum NickelPlated Needle Assembly Bearings PEEK Thermal Control Body 6061-T6Aluminum Nickel Plated Reservoir Holder Acetyl Reservoir Size 5, 10, or30 cc (0.17, 0.34, or 1.0 oz) Feed Tube Assembly Fitting Female Luer perANSI/HIMA MD70.1-1983 Maximum Cycle Frequency 200 Hz. Minimum Valve AirPressure 5.5 bar (80 psi) Operating Noise Level 70 db* Solenoid 24 VDC,12.7 Watts Thermal Control Heater 24 VDC, 14.7 Watts, 40 ohms ThermalControl RTD 100 ohm, platinum Maximum Heater Set Point 80 C. *At MaximumCycle Rate

An exploded view of this apparatus is shown in FIG. 54B.

Theory of Operation of DJ-9000

The DJ-9000 has a normally closed, air-actuated, spring-returnmechanism, which uses momentum transfer principles to expel precisevolumes of material. Pressurized air is regulated by a high-speedsolenoid to retract a needle assembly from the seat. Fluid, fed into thefluid chamber, flows over the seat. When the air is exhausted, theneedle travels rapidly to the closed position, displacing fluid throughthe seat and nozzle in the form of a droplet. Multiple droplets fired insuccession can be used to form larger dispense volumes and lines whencombined with the motion of a dispenser robot.

The equipment has various adjustable features: The following featuresaffect performance of the DJ-9000 and are typically adjusted to fitspecific process conditions.

Fluid Pressur should be set so that fluid fills to the seat, but shouldnot be influential in pushing the fluid through the seat and nozzle. Ingeneral, higher fluid pressure results in a larger volume of materialjetted.

The Stroke Adjustment controls the travel distance of the NeedleAssembly. The control is turned counterclockwise to increase needleassembly travel, or turned clockwise to decrease travel. An increase oftravel distance will often result in a larger volume of material jetted.

The Solenoid Valve controls the valve operation. When energized, itallows air in the jet air chamber to compress a spring and thereby raisethe Needle Assembly. When de-energized, the air is released and thespring forces the piston down so that the needle tip contacts the seat.

The seat and nozzle geometry are typically the main factors controllingdispensed material volume. The seat and nozzle size are determined basedon the application and fluid properties. Other parameters are adjustedin accordance with seat and nozzle choices. Available seat and nozzlesizes are listed in the table hereinbelow.

Thermal Control Assembly: Fluid temperature often influences fluidviscosity and flow characteristics. The DJ-9000 is equipped with aThermal Control Assembly that assures a constant fluid temperature.

Dot and Line Parameters: In addition to the DJ-9000 hardwareconfiguration and settings, Dot and Line Parameters are set in asoftware program (referred to as FmNT) to control the size and qualityof dots and lines dispensed.

Wax Loading in Valves

FIGS. 55A and 558 show how a combination of controlled hot dropdispensing into a heated microchannel device of the right dimensions andgeometry is used to accurately load wax into a microchannel of amicrofluidic cartridge to form a valve. The heated dispenser head can beaccurately position over an inlet hole of the microchannel in themicrofluidic device, and can dispense molten wax drops in volumes assmall as 75 nanoliters with an accuracy of 20%. The inlet hole of themicrochannel device is dimensioned in such a way that the droplet of 75nl can be accurately shot to the bottom of the inlet hole using, forexample, compressed air, or in a manner similar to an inkjet printingmethod. The microchannel device is maintained at a temperature above themelting point of the wax thereby permitting the wax to stay in a moltenstate immediately after it is dispensed. After the drop falls to thebottom of the inlet hole, the molten wax is drawn into the narrowchannel by capillary action. The volume of the narrow section isdesigned to be approximately equal to a maximum typical amount that isdispensed into the inlet hole.

Heater Multiplexing (Under Software Control)

Another aspect of the apparatus described herein, relates to a methodfor controlling the heat within the system and its components, asillustrated in FIG. 56. The method leads to a greater energy efficiencyof the apparatus described herein, because not all heaters are heatingat the same time, and a given heater is receiving current for only pertof the time.

Generally, the heating of microfluidic components, such as a PCRreaction zone, is controlled by passing currents through suitablyconfigured microfabricated heaters. The heating can be furthercontrolled by periodically turning the current on and off with varyingpulse width modulation (PWM), wherein pulse width modulation refers tothe on-time/off-time ratio for the current. The current can be suppliedby connecting a microfabricated heater to a high voltage source (forexample, 30V), which can be gated by the PWM signal. In someembodiments, the device includes 48 PWM signal generators. Operation ofa PWM generator includes generating a signal with a chosen, programmableperiod (the end count) and granularity. For instance, the signal can be4000 μs (micro-seconds) with a granularity of 1 us, in which case thePWM generator can maintain a counter beginning at zero and advancing inincrements of 1 μs until it reaches 4000 μs, when it returns to zero.Thus, the amount of heat produced can be adjusted by adjusting the endcount. A high end count corresponds to a greater length of time duringwhich the microfabricated heater receives current and therefore agreater amount of heat produced.

In various embodiments, the operation of a PWM generator can alsoinclude a programmable start count in addition to the aforementioned endcount and granularity. In such embodiments, multiple PWM generators canproduce signals that can be selectively non-overlapping (e.g., bymultiplexing the on-time of the various heaters) such that the currentcapacity of the high voltage power is not exceeded. Multiple heaters canbe controlled by different PWM signal generators with varying start andend counts. The heaters can be divided into banks, whereby a bankdefines a group of heaters of the same start count. For example, 36 PWMgenerators can be grouped into six different banks, each correspondingto a certain portion of the PWM cycle (500 ms for this example). The endcount for each PWM generator can be selectively programmed such that notmore than six haters will be on at any given time. A portion of a PWMcycle can be selected as dead time (count 3000 to 4000 for this example)during which no heating takes place and sensitive temperature sensingcircuits can use this time to sense the temperature. The table belowrepresents a PWM cycle for the foregoing example:

Start Count End Count Max End count Bank 1 PWM generator#1 0 150 500 PWMgenerator#2 0 220 500 . . . . . . . . . PWM generator#6 0 376 500 Bank 2PWM generator#7 500 704 1000 PWM generator#8 500 676 1000 . . . . . . .. . . . . PWM generator#12 500 780 1000 Bank 3 PWM generator#13 10001240 1500 PWM generator#14 1000 1101 1500 . . . . . . . . . . . . PWMgenerator#18 1000 1409 1500 Bank 4 PWM generator#19 1500 1679 2000 PWMgenerator#20 1500 1989 2000 . . . . . . . . . . . . PWM generator#241500 1502 2000 Bank 5 PWM generator#25 2000 2090 2500 PWM generator#262000 2499 2500 . . . . . . . . . . . . PWM generator#30 2000 2301 2500Bank 6 PWM generator#31 2500 2569 3000 PWM generator#32 2500 2790 3000 .. . . . . . . . . . . PWM generator#36 2500 2678 3000

Use of Detection System to Measure/Detect Fluid in PCR Chamber

The apparatus optionally has a very sensitive fluorescence detector thatis able to collect fluorescence light from the PCR chamber 210 of amicrofluidic cartridge. This detector is used to detect the presence orliquid in the chamber, a measurement that determines whether or not tocarry out a PCR cycle. A background reading is taken prior to fillingthe chamber with liquid. Another reading is taken after microfluidicoperations have been performed that should result in filling the PCRchamber with liquid. The presence of liquid alters the fluorescencereading from the chamber. A programmable threshold value is used to tunean algorithm programmed into the processor (for example, the secondreading has to exceed the first reading by 20%). If the two readings donot differ beyond the programmed margin, the liquid is deemed to nothave entered the chamber, and a PCR cycle is not initiated for thatchamber. Instead, a warning is issued to a user.

Computer Program Product

In various embodiments, a computer program product for use with theapparatus herein includes computer readable instructions thereon foroperating the apparatus.

In various embodiments, the computer program product can include one ormore instructions to cause the system to: output an indicator of theplacement of the microfluidic cartridge in the bay; read a sample labelor a microfluidic cartridge label; output directions for a user to inputa sample identifier, output directions for a user to load a sampletransfer member with the PCR-ready sample; output directions for a userto introduce the PCR-ready sample into the microfluidic cartridge;output directions for a user to place the microfluidic cartridge in thereceiving bay; output directions for a user to close the lid to operatethe force member; output directions for a user to pressurize thePCR-ready sample in the microfluidic cartridge by injecting thePCR-ready sample with a volume of air between about 0.5 mL and about 5mL; and output status information for sample progress from one or morelanes of the cartridge.

In various embodiments, the computer program product can include one ormore instructions to cause the system to: heat the PCR ready-sampleunder thermal cycling conditions suitable for creating PCR ampliconsfrom the neutralized polynucleotide; contact the neutralizedpolynucleotide sample or a PCR amplicon thereof with at least one probethat is selective for a polynucleotide sequence; independently contacteach of the neutralized polynucleotide sample and a negative controlpolynucleotide with the PCR reagent mixture under thermal cyclingconditions suitable for independently creating PCR amplicons of theneutralized polynucleotide sample and PCR amplicons of the negativecontrol polynucleotide; contact the neutralized polynucleotide sample ora PCR amplicon thereof and the negative control polynucleotide or a PCRamplicon thereof with at least one probe that is selective for apolynucleotide sequence; output a determination of the presence of apolynucleotide sequence in the biological sample, the polynucleotidesequence corresponding to the probe, if the probe is detected in theneutralized polynucleotide sample or a PCR amplicon thereof; and/oroutput a determination of a contaminated result if the probe is detectedin the negative control polynucleotide or a PCR amplicon thereof.

In various embodiments, the computer program product can include one ormore instructions to cause the system to automatically conduct one ormore of the steps of the method.

In various embodiments, the microfluidic cartridge comprises two or moresample lanes, each including a sample inlet valve, a bubble removalvent, a thermally actuated pump, a thermally actuated valve, and a PCRreaction zone, wherein the computer readable instructions are configuredto independently operate one or more components of each said lane in thesystem, independently of one another, and for causing a detector tomeasure fluorescence from the PCR reaction zones.

Sample

In various embodiments, the sample can include a PCR reagent mixturecomprising a polymerase enzyme and a plurality of nucleotides. The PCRreagent mixture can be in the form of one or more lyophilized pelletsand the steps by which the PCR-ready sample is prepared can involvecontacting the PCR pellet with liquid to create a PCR reagent mixturesolution. In yet another embodiment, each of the PCR lanes may havedried down or lyophilized ASR reagents preloaded such that the user onlyneeds to input prepared polynucleotide sample into the PCR. In anotherembodiment, the PCR lanes may have only the application-specific probesand primers premeasured and preloaded, and the user inputs a samplemixed with the PCR reagents.

In various embodiments, the microfluidic network can be configured tocouple heat from an external heat source to a sample mixture comprisingPCR reagent and neutralized polynucleotide sample under thermal cyclingconditions suitable for creating PCR amplicons from the neutralizedpolynucleotide sample.

In various embodiments, the PCR ready sample can further include apositive control plasmid and a fluorogenic hybridization probe selectivefor at least a portion of the plasmid. In various embodiments, thePCR-ready sample further includes a sample buffer, and at least oneprobe that is selective for a polynucleotide sequence, e.g., thepolynucleotide sequence that is characteristic of a pathogen selectedfrom the group consisting of gram positive bacteria, gram negativebacteria, yeast, fungi, protozoa, and viruses.

In various embodiments, the microfluidic cartridge can accommodate anegative control polynucleotide, wherein the microfluidic network can beconfigured to independently carry out PCR on each of a neutralizedpolynucleotide sample and a negative control polynucleotide with the PCRreagent mixture under thermal cycling conditions suitable forindependently creating PCR amplicons of the neutralized polynucleotidesample and PCR amplicons of the negative control polynucleotide. Eachlane of a multi-lane cartridge as described herein can perform tworeactions because of the presence or two fluorescence detection systemsper lane. A variety of combinations of reactions can be performed in thecartridge, such as two sample reactions in one lane, a positive controland a negative control in two other lanes; or a sample reaction and aninternal control in one lane and a negative control in a separate lane.

In various embodiments, the sample can include at least one probe thatcan be selective for a polynucleotide sequence, wherein the steps bywhich the PCR-ready sample is prepared involve contacting theneutralized polynucleotide sample or a PCR amplicon thereof with theprobe. The probe can be a fluorogenic hybridization probe. Thefluorogenic hybridization probe can include a polynucleotide sequencecoupled to a fluorescent reporter dye and a fluorescence quencher dye.The PCR reagent mixture can further include a positive control plasmidand a plasmid fluorogenic hybridization probe selective for at least aportion of the plasmid and the microfluidic cartridge can be configuredto allow independent optical detection of the fluorogenic hybridizationprobe and the plasmid fluorogenic hybridization probe.

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of an organism, for example any organismthat employs deoxyribonucleic acid or ribonucleic acid polynucleotides.Thus, the probe can be selective for any organism. Suitable organismsinclude mammals (including humans), birds, reptiles, amphibians, fish,domesticated animals, wild animals, extinct organisms, bacteria, fungi,viruses, plants, and the like. The probe can also be selective forcomponents of organisms that employ their own polynucleotides, forexample mitochondria. In some embodiments, the probe is selective formicroorganisms, for example, organisms used in food production (forexample, yeasts employed in fermented products, molds or bacteriaemployed in cheeses, and the like) or pathogens (e.g., of humans,domesticated or wild mammals, domesticated or wild birds, and the like).In some embodiments, the probe is selective for organisms selected fromthe group consisting of gram positive bacteria, gram negative bacteria,yeast, fungi, protozoa, and viruses.

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of an organism selected from the groupconsisting of Staphylococcus spp., e.g., S. epidermidis, S. aureus,Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistantStaphylococcus; Streptococcus (e.g., α, β or γ-hemolytic, Group A, B, C,D or G) such as S. pyogenes, S. agalactiae; E. faecalis, E. durans, andE. faecium (formerly S. faecalis, S. durans, S. faecium);nonenterococcal group D streptococci. e.g., S. bovis and S. equines;Sueptococci viridans, e.g., S. mutans, S. sanguis, S. salivarius, S.mitior, A. milleri, S. constellatus, S. intermedius, and S. anginosus;S. iniae; S. pneumoniae; Neisseria, e.g., N. meningitides. N.gonorrhoeae, saprophytic Neisseria sp; Erysipelothrix, e.g., E.rhusiopathias; Listeria spp., e.g., L. monocytogcncs, rarely L. ivanoviiand L. sceligei; Bacillus, e.g., B. anthracis, B. cerus, B. subtilis, B.subtilus niger, B. huringiensis; Nocardia asteroids; Legionella, e.g.,L. pnecumonophilia, Pneumocystis. e.g., P. carinii; Enterobcteriaceasuch as Salmonella, Shigella, Escherichia (e.g., E. coli, E. coliO157:H7); Klebsiella, Enterobacter, Serratia, Proteus, Morganella,Providencia, Yersinia, and the like. e.g., Salmonella, e.g., S. typhi S.paratyphi A, B (S. schottmuelleri), and C (S. hirschrldii), S. dublin S.choleraesuis, S. cnteritidis, S. typhimurium, S. heidelberg, S. newport,S. infantis, S. agona, S. montevideo, and S. sint-paul; Shigella e.g.,subgroups: A. B, C, and D, such as S. flexneri, S. sonnei, S. boydii, S.dysenteria; Proteus (P. mirabilis, P. vulgaris, and P. myxofaciens),Morganella (M. morganii); Providencia (P. rettgeri, P. alcalifaciens,and P. stuartii); Yersinia, e.g., Y. pestis, Y. encerocolitica;Haemophilus, e.g., H. influenzae, H. perainfluenzae H, aphrophilus, H.ducrcyi; Brucella, e.g., B. abortus B. melitensis, B. suis, B. canis;Francisella, e.g., F. tularensis; Pseudomonas, e.g., P. araginosa, P.paucimobilis, P. putida, P. fluorescens. P. acidovorans, Burkholderia(Pseudomonas) pseudomallei, Burkholderia mallei, Burkholderia cepaciaand Stenotrophomonas mallophilia; Campylobacter, e.g., C. fetus fetus,C. jejuni, C. pylori (Helicobacter pylori); Vibrio. e.g., V. cholerae,V. parahaemolyticus. V. mimicus, V. alginolyticus, V. hollisase, V.vulnificus, and the nonagglutinable vibrios; Clostridia, e.g., C.perfringens, C. tetani, C. difficile, C. botulinum; Actinomyces, e.g.,A. israelii; Bacteroides, e.g., B. fragilis, B. thetaiotaomicon, B.distasonis, B. vulgatus, B. ovatus, B. caccae, and B. merdae;Prevotella, e.g., P. melaninogenica; genus Fusobacterium; Treponema,e.g. T. pallidum subspecies endemicum, T. pallidum subspecies pertnue,T. carateum, and T. pallidum subspecies pallidum; genus Borrelia, e.g.,B burgdorferi; genus Leptospira; Streptobacillus, e.g., S. moniliformis;Spirillum, e.g., S. minus; Mycobacterium, e.g., M. tuberculosis. M.bovis, M. africanum, M. avium M. intracellulare, M. kansasii, M. xenopi,M. marinum, M. ulcerans, the M. fortuitum complex (M. fortuitum and M.chelonci), M. leprac, M. asiaticum, M. chelonci subsp. abscessus, M.fallax, M, fortuitum, M. malmoenac, M. shimoidei, M. simiae, M. szulgai,M. xenopi; Mycoplasma, e.g., M. hominis, M. orale, M. salivarium, M.fermentans, M. pneumoniae, M. bovis, M. tuberculosis, M. avium, M.leprae; Mycoplasma. e.g., M. genitalium; Ureaplasma, e.g., U.uroalyticum; Trichomonas, e.g., T. vaginalis; Cryptococcus, e.g., C.neoformans; Hissoplasma, e.g., H. capsulatum; Candida, e.g., C.albicans; Aspergillus sp; Coccidioides, e.g., C. immitis; Blastomyces,e.g. B. dermatildis; Paracoccidioides, e.g., P. brasiliensis;Penicillium, e.g., P. marneffei; Sporothrix, e.g., S. schenckii;Rhizopus, Rhizomucor, Absidia, and Basidiobolus; diseases caused byBipolaris, Cladophialophora, Cladosporium, Drechslera. Exophiala,Fonsecara, Phialophora, Xylohypha, Ochroconis, Rhinocladiella,Scolecobasidium, and Wangiella; Trichosporon, e.g., T. beiglii;Blastoschizomyces, e.g., B. capitatus; Plasmodium, e.g., P. aklcipanum,P. vivax, P. ovale, and P. malariae; Babesia sp; protozoa of the genusTrypenosoms, e.g., T. cnrui; Leishmania. e.g., L. donovani, L. major L.tropica, L. mexicana, L. braziliensas, L. viannia braziliansis;Toxoplasma, e.g., T. gondii; Amoebas of the genera Naegleria orAcanthamoebs; Entamnoeb histolytica; Oiardia lamblia; genusCryptosporidium, e.g., C. parvum; Isospora belli; Cyclosporacayetanensis; Ascaris lumbricoides; Trichuris trichium; Ancylostomaduodenale or Necator americanus; Strongyloide stercoralis Toxocara,e.g., T. canis, T. cati; Baylisascaris, e.g., B. procyonis; Trichinella,e.g., T. spiralis; Dracunculus, e.g., D. medinensis; genus Filarioidea;Wuchereria bancrofti; Brugia, e.g., B. malayi, or B. timori; Onchocercavolvulus; Loa loa; Dirofilaria immitis; genus Schistosoma, e.g., S.japonicum, S. mansoni, S. mekongi, S intercalatum, S. haematobium;Paragonimus, e.g., P. Westermani, P. Skriabini; Clonorchis sinensis;Fasciola hepatica; Opisthorchis sp; Fasciolopsis buski; Diphyllobothriumlatum; Tacnia, e.g., T. saginaw, T. solium; Echinococcus, e.g., E.granulosus, E. muhiloculuris; Picornaviruses, rhinoviruses echoviruses,coxsackieviruses, influenza virus; paramyxoviruses, e.g., types 1, 2, 3,and 4; adenoviruses; Herpesviruses, e.g., HSV-1 and HSV-2;varicella-zoster virus; human T-lymphotrophic virus (type I and typeII); Arboviruses and Arenaviruses; Togaviridae, Flaviviridac,Bunyaviridae, Reoviridac; Flavivirus; Hantavirus; Viral encephalitis(alphaviruses (e.g., Venezuclan equine encephalitis, castern equineencephalitis, western equine encephalitis]); Viral hemorrhagic fevers(filoviruses. [e.g., Ebola, Marburg] and arnaviruses (e.g., Lana,Machupo)); Smallpox (variola); retroviruses e.g., human immunodeficiencyviruses 1 and 2; human papillomavirnas [HPV] types 6, 11, 16, 18, 31.33,and 35.

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of an organisms selected from the groupconsisting of Pseudomonas aeruginaa, Proteus mirabilis, Klebstellaaxytoca, Klebsiella pneumoniae, Escherichiu coli, AcinelobaeterSaumannii, Serratia marcescens, Enterobacter aerogenes, Enterococcusfaecium, vancomycin-resistant Enterococcus (VRE), Staphylococcus aureus,methecillin-resistant Staphylococcus aureus (MRSA), Streptococcusviridans, Listeria nonocytogenes, Enterococcus spp., Streptococcus GroupB, Streptococcus Group C, Streptococcus Group G, Streptococcus Group F,Enterococcus faecalis, Streptococcus pneumoniae, Staphylococcusepidermidis, Gardenerella vaginalis, Micrococcus sps., Haemophilusinfluenzae, Neisseria gonorrhoeee, Moraxello cutarrahlis, Salmonellasps., Chlamydia trachomatii, Pepostrepiococcus productus,Pepostreptococcus anaerobius, Lactibacillus fermentum, Eubaceriumlentum, Candida glabraua, Candida albicans, Chlamydia spp., Campiobacterspp., Salmonella spp., smallpox (variola major), Yersina Pestis, HerpesSimplex Virus I (HSV I), and Herpes Simplex Virus II (HSV II).

In various embodiments, the probe can be selective for a polynucleotidesequence thait is characteristic of Group B Streptococcus.

Carrying out PCR on a PCR-ready sample can include heating the PCRreagent mixture and the neutralized polynucleotide sample under thermalcycling conditions suitable for creating PCR amplicons from theneutralized polynucleotide sample; contacting the neutralizedpolynucleotide sample or a PCR amplicon thereof with at least one probethat is selective for a polynucleotide sequence; independentlycontacting each of the neutralized polynucleotide sample and a negativecontrol polynucleotide with the PCR reagent mixture under thermalcycling conditions suitable for independently creating PCR amplicons ofthe neutralized polynucleotide sample and PCR amplicons of the negativecontrol polynucleotide; and/or contacting the neutralized polynucleotidesample or a PCR amplicon thereof and the negative control polynucleotideor a PCR amplicon thereof with at least one probe that is selective fora polynucleotide sequence.

In various embodiments, a method of carrying out PCR on a sample canfurther include one or more of the following steps: heating thebiological sample in the microfluidic cartridge; pressurizing thebiological sample in the microfluidic cartridge at a pressuredifferential compared to ambient pressure of between about 20kilopascals and 200 kilopascals, or in some embodiments between about 70kilopascals and 110 kilopascals.

In various embodiments, a method of using the apparatus described hereincan further include one or more of the following steps: determining thepresence of a polynucleotide sequence in the biological sample, thepolynucleotide sequence corresponding to the probe, if the probe isdetected in the neutralized polynucleotide sample or a PCR ampliconthereof; determining a contaminated result if the probe is detected inthe negative control polynucleotide or a PCR amplicon thereof; and/or insome embodiments, wherein the PCR reagent mixture further comprises apositive control plasmid and a plasmid probe selective for at least aportion of the plasmid, the method further including determining a PCRreaction has occurred if the plasmid probe is detected.

Fluorescence Detection System. Including Lense and Filters, and MultipleParallel Detection for a Multi-Lane Cartridge

A miniaturized, highly sensitive fluorescence detection system can beincorporated for monitoring fluorescence from the biochemical reactionsthat are the basis of nucleic acid amplification methods such as PCR.

Accordingly, another aspect of the apparatus includes a system formonitoring fluorescence from biochemical reactions. The system can be,for example, an optical detector having a light source (for example anLED) that selectively emits light in an absorption band of a fluorescentdye, lenses for focusing the light, and a light detector (for example aphotodiode) that selectively detects light in an emission band of thefluorescent dye, wherein the fluorescent dye corresponds to afluorescent polynucleotide probe or a fragment thereof. Alternatively,the optical detector can include a bandpass-filtered diode thatselectively emits light in the absorption band of the fluorescent dye (afluorogenic probe) and a bandpass filtered photodiode that selectivelydetects light in the emission band of the fluorescent dye. For example,the optical detector can be configured to independently detect aplurality of fluorescent dyes having different fluorescent emissionspectra, wherein each fluorescent dye corresponds to a fluorescentpolynucleotide probe or a fragment thereof. For example, the opticaldetector can be configured to independently detect a plurality offluorescent dyes at a plurality of different locations of, for example,a microfluidic cartridge, wherein each fluorescent dye corresponds to afluorescent polynucleotide probe or a fragment thereof.

In some embodiments, a given detector for use with the apparatusdescribed herein is capable of detecting a fluorescence signal fromnanoliter scale PCR reactions. Advantageously, the detector is formedfrom inexpensive components, having no moving parts. The detector isalso configured to mate with a microfluidic cartridge as furtherdescribed herein, and is also preferably part of a pressure applicationsystem, such as a sliding lid, that keeps the cartridge in place. Thedetector further has potential for 2 or 3 color detection and iscontrolled by software, preferably custom software, configured to sampleinformation from the detector.

FIGS. 57-59 depict an embodiment of a highly sensitive fluorescencedetection system including light emitting diodes (LED's), photodiodes,and filters/lenses for monitoring, in real-time, one or more fluorescentsignals emanating from the microfluidic cartridge. The embodiment inFIGS. 57-59 has a two-color detection system having a modular designthat mates with a single lane microfluidic cartridge. The detectorcomprises two LED's (blue and red, respectively) and two photodiodes.The two LED's are configured to transmit a beam of focused light on to aparticular region of the cartridge. The two photodiodes are configuredto receive light that is emitted from the region of the cartridge. Onephotodiode is configured to detect emitted red light, and the otherphotodiode is configured to detect emitted blue light.

FIGS. 60 and 61 show an exemplary read-head comprising a multiplexed 2color detection system, such as multiple instances of a detection systemshown in FIGS. 57-59, that is configured to mate with a multi-lanemicrofluidic cartridge. FIG. 60 shows a view of the exterior of amultiplexed read-head. FIG. 61 is an exploded view that shows howvarious detectors are configured within an exemplary multiplexed readhead, and in communication with an electronic circuit board.

The module in FIGS. 60 and 61 is configured to detect fluorescence fromeach lane of a 12-lane cartridge, and therefore comprises 24independently controllable detectors, arranged as 12 pairs of identicaldetection elements. Each pair of elements is then capable of dual-colordetection of a pre-determined set of fluorescent probes. It would beunderstood by one of ordinary skill in the art that other numbers ofpairs of detectors are consistent with the apparatus described herein.For example, 4, 6, 8, 10, 16, 20, 24, 25, 30, 32, 36, 40, and 48 pairsare also consistent and can be configured according to methods andcriteria understood by one of ordinary skill in the art.

Exemplary Optics Assembly

In an exemplary embodiment, the optical chassis/pressure assembly ishoused in an enclosure (made of plastic in certain embodiments) that canbe positioned to cover a multi-lane microfluidic cartridge. Theenclosure can optionally have a handle that can be easily grasped by auser, and is guided for smooth and easy pushing and pulling. The handlemay also serves as a pressure-locking device. The enclosure's horizontalposition is sensed in both the all-open and in the all-forward position,and reported to controlling software. The enclosure and optical chassispressure assembly registers with a heater cassette module positionedunderneath a microfluidic cartridge to within 0.010″. A close ft isimportant for proper heater/cartridge interface connections. Theenclosure assembly does not degrade in performance over a life of 10,000cycles, where a cycle is defined as: beginning with the slider in theback position, and sliding forward then locking the handle down on acartridge, unlocking the handle and returning it to the original backposition. All optical path parts should be non-reflective (anodized,painted, molded, etc.) and do not lose this feature for 10,000 cycles.The optics unit is unaffected by a light intensity of <=9,000root-candles from a source placed 12″ from the instrument at angleswhere light penetration is most likely to occur. No degradation ofperformance is measured at the photo-detector after 10,000 cycles.

When fabricating a detector assembly, a single channel is made thathouses two LED sources (blue and amber) and two additional channels thathouse one photodiode detector each (four total bored holes). The twopaired channels (source and detector) are oriented 430 from each other,measured from the optical axis and are in-line with the other pairedchannels that are at the same 43° orientation. The holes bored in theoptical chassis contain filters and lenses with appropriate spacers, thespecifications of which are further described herein. The LED's are heldin place to prevent movement as the mechanical alignment is importantfor good source illumination. The LED's are preferably twisted until thetwo “hot spots” are aligned with the reading channels on the cartridge.This position must be maintained until the LED's cannot be moved. Theoptical chassis can be made of aluminum and be black anodized. Thebottom pressure surface of the optical chassis is flat to ±0.001″ acrossthe entire surface. The optical chassis is center-balanced such that thecenter of the optical chassis force is close to the center of thereagent cartridge. The pressure assembly (bottom of the optical chassis)provides uniform pressure of a minimum of 1 psi across all heatersections of the reagent cartridge. The optical assembly can be movedaway from the reagent cartridge area for cartridge removal andplacement. Appropriate grounding of the optical chassis is preferred toprevent spurious signals to emanate to the optic PCB.

The LED light sources (amber and blue) are incident on a microfluidiccartridge through a bend pass filter and a focusing lens. These LEDlight sources have a minimum output of 2800 millicandles (blue) and 5600millicandles (Green), and the center wavelengths are 470 (blue) and 575(amber) nanometers, with a half band width of no more than 75nanometers.

The LED light excites at least one fluorescent molecule (initiallyattached to an oligonucleotide probe) in a single chamber on acartridge, causing it to fluoresce. This fluorescence will normally beefficiently blocked by a closely spaced quencher molecule. DNAamplification via TAQ enzyme will separate the fluorescent and quenchingmolecules from the oligonucleotide probe, disabling the quenching. DNAamplification will only occur if the probe's target molecule (a DNAsequence) is present in the sample chamber. Fluorescence occurs when acertain wavelength strikes the target molecule. The emitted light is notthe same as the incident light. Blue incident light is blocked from thedetector by the green only emission filter. Green incident lightsimilarly is blocked from the detector by the yellow emission filter.The fluorescent light is captured and travels via a pathway into afocusing lens, through a filter and onto a very sensitive photodiode.The amount of light detected increases as the amount of the DNAamplification increases. The signal will vary with fluorescent dye used,but background noise should be less than 1 mV peak-to-peak. Thephoto-detector, which can be permanently mounted to the optical chassisin a fixed position, should be stable for 5 years or 10,000 cycles, andshould be sensitive to extremely low light levels, and have a dark valueof no more than 60 mV. Additionally, the photo-detector must becommercially available for at least 10 years. The lenses arePlano-convex (6 mm detector, and 12 mm source focal length) with theflat side toward the test cartridge on both lenses. The filters shouldremain stable over normal operating humidity and temperature ranges.

The filters, e.g., supplied by Omega Optical (Brattleboro, Vt. 05301),are a substrate of optical glass with a surface quality of F/F perMil-C-48497A. The individual filters have a diameter of 6.0±0.1 mm, athickness of 6.0±0.1 mm, and the AOI and ½ cone AOI is 0 degrees and ±8degrees, respectively. The clear aperture is >/=4 mm diameter and theedge treatment is blackened prior to mounting in a black, anodized metalring. The FITC exciter filters is supplied by, e.g., Omega Optical (PN481AF30-RED-EXC). They have a cut-off frequency of 466±4 nm and a cut-onfrequency of 496±4 nm. Transmission is >/=65% peak and blockingis: >/=OD8 in theory from 503 to 580 nm, >/=OD8 from 501-650 nm, >/=OD4avg. over 651-1000 nm, and >/=OD4 UV-439 nm. The FITC emitter filters issupplied by, e.g., Omega Optical (PN 534AF40-RED-EM). They will have acut-off frequency of 514±2 nm and a cut-on frequency of 554±4 nm.Transmission is >/=70% peak and blocking is: >/=OD5 in theory from 400to 504 nm, >/=OD5 UV-507 nm, and >/=OD4 avg, 593-765 nm. The amberexciter filters are supplied by, e.g., Omega Optical (PN582AF25-RED-EXC). They have a cut-off frequency of 594±5 nm and a cut-onfrequency of 569±5 nm. Transmission is >/=70% peak and blockingis: >/=OD5 in theory from 600 to 700 nm, >/=OD5 600-900 um, and >/=OD4UV-548 nm. The amber emitter filters are supplied by, e.g., OmegaOptical (PN 627AF30-RED-EM). They have a cut-off frequency of 642±5 nmand a cut-on frequency of 612±5 nm. Transmission is >/=70% peak andblocking is: >/=OD5 in theory from 550 to 600 μm, >/=OD5 UV-605 nm,and >/=OD5 avg, 667-900 nm. The spacers should be inert and temperaturestable throughout the entire operating range and should maintain thefilters in strict position and alignment. The epoxy used should haveoptically black and opaque material and dry solid with no tacky residue.Additionally, it should have temperature and moisture stability, exertno pressure on the held components, and should mount the PCB in such away that it is fixed and stable with no chances of rotation or verticalheight changes, 50% of illumination shall fall on the sample planewithin an area 0.1″ (2.5 mm) wide by 0.3″ (7.5 mm) along axis of thedetection channel. Fluorescence of the control chip should not changemore than 0.5% of the measured signal per 0.001″ of height though aregion±0.010 from the nominal height of the control chip.

An exemplary optics board is shown in FIG. 62, and is used to detect andamplify the fluorescent signature of a successful chemical reaction on amicro-fluidic cartridge, and controls the intensity of LED's usingpulse-width modulation (PWM) to illuminate the cartridge sample over upto four channels, each with two color options. Additionally, it receivesinstructions and sends results data back over an LVDS (low-voltagedilfferential signaling) SPI (serial peripheral interface). The powerboard systems include: a +12V input; and +3.3V, +3.6V, +5V, and −5Voutputs, configured as follows: the +3.3V output contains a linearregulator, is used to power the LVDS interface, should maintain a +/−5%accuracy, and supply an output current of 0.35 A; the +3.6V outputcontains a linear regulator, is used to power the MSP430, shouldmaintain a +/=5% accuracy, and supply an output current of 0.35 A; the+5V output contains a linear regulator, is used to power the plus railfor op-amps, should maintain a +/=5% accuracy, and supply an outputcurrent of 0.35 A; the −5V output receives its power from the +5Vsupply, is used to power the minus rail for op-amps and for thephoto-detector bias, should maintain a +/=1% voltage accuracy, andsupply an output current of 6.25 mA+1-10%. Additionally, the power boardhas an 80 ohm source resistance, and the main board software canenable/disable the regulator outputs.

The main board interface uses a single channel of the LVDS standard tocommunicate between boards. This takes place using SPI signaling overthe LVDS interface which is connected to the main SPI port of thecontrol processor. The interface also contains a serial port forin-system programming.

The exemplary optical detection system of FIG. 62 consists of a controlprocessor, LED drivers, and a photo-detection system. In the exemplaryembodiment, the control processor is a TI MSP430F1611 consisting of adual SPI (one for main board interface and one for ADC interface) andextended SRAM for data storage. It has the functions of powermonitoring. PWM LED control, and SPI linking to the ADC and main board.The LED drivers contain NPN transistor switches, are connected to thePWM outputs of the control processor, can sink 10 mA @12V per LED (80 mAtotal), and are single channel with 2 LEDs (one of each color) connectedto each. The photo-detection system has two channels and consists of aphoto-detector, high-sensitivity photo-diode detector, high pin currentto voltage converter, unity pin voltage inverting amplifier, and an ADC.Additionally it contains a 16 channel Sigma-delta (only utilizing thefirst 8 channels) which is connected to the second SPI port of thecontrol processor. It would be understood by one of ordinary skill inthe art that other choices and combinations of elements can be broughttogether to make a functioning detection system consistent with thedescription herein.

Additional Advantages and Features of the Technology Herein

The use of a disposable process chamber, having surface coating andmaterial properties to allow low volume, and open tube heated release tomaximize sample concentration in lowest volume possible.

The integrated magnetic heat separator that allows multiple samples tobe heated independently but separated using a single moveable magnetplatform.

A reader/tray design that allows easy placement of microfluidiccartridge and multiple sample pipetting of liquid using a roboticdispenser in one position; relative displacement to another location andpressure application for subsequent rapid heat incubation steps andoptical detection. The bottom surface of the cartridge mates with theheating surface. Furthermore, it is typically easier to move a cartridgeand heater in and out of position than a detector.

A moveable readhead design for fluorescence detection from microfluidicPCR channels.

Aspects of the holder, such as a unitized disposable strip, that includethe presence of sealed lyophilized reagents as well as liquids sealed inclose proximity, which is normally hard to achieve. The laminatesdeployed herein make storage easier.

The holder permits snapping of multiple ASR tubes, and associated liquiddispensing processes that minimizes cross-sample contamination butmultiple PCR preparations to be performed from a single clinical sample.

Software features allow a user to either get results from all 24 samplesas quickly as possible or the first 12 samples as quickly as possibleand the next 12 later.

The preparatory and diagnostic instruments described herein enablesdifferent sample types (such as blood, urine, swab, etc.) to be allprocessed at the same time even though each may require differenttemperatures, times or chemical reagents. This is achieved in part byusing individualized but compatible holders.

Automatic feeding of microfluidic cartridges into a PCR reader via acartridge autoloader saves a user time and leads to increased efficiencyof overall operation.

Piercing through foil over a liquid tube and reliable way of picking upliquid.

A moveable read-head that has the pumps, sensors (pipette detection,force sensing), sample identification verifier, etc., moving with it,and therefore minimizes the number of control lines that move across theinstrument during use.

Accurate and rapid alignment of pipette tips with cartridge inlet holesusing a motorized alignment plate.

EXAMPLES Example 1: Reagent Holder

An exemplary reagent holder consistent with the description herein hasthe following dimensions and capacities:

-   -   180 mm long×22 mm wide×100 mm tall;    -   Made from Polypropylene.    -   One snapped-in low binding 1.7 ml tube that functions as a        process tube.    -   3 built-in tubes that function as receptacles for reagents, as        follows:        -   One tube containing 200-1000 μl of wash buffer (0.1 mM Tris,            pH 8).        -   One tube containing 200-1000 μl of release solution (40 mM            NaOH).        -   One tube containing 200-1000 μl of neutralization solution            (330 mM Tris, pH 8.0).    -   One built-in tube that functions as a waste chamber (will hold        ˜4 ml of liquid waste).    -   3 receptacles to accept containers for solid reagents. Snap-in        0.3 ml or 0.65 ml PCR tubes (which are typically stored        separately from the reagent holder) are placed in each of these        locations, and contain, respectively:        -   lyophilized sample preparation reagents (lysis enzyme mix            and magnetic affinity beads).        -   First lyophilized PCR master mix, probes and primers for a            first target analyte detection.        -   Second lyophilized PCR master mix, probes and primers for a            second target analyte detection (only offered in select            cases, such as detection of Chlamydia and Gonorrhea from            urine).    -   4 pipette tips located in 4 respective sockets.    -   Pipette tip Sheath: The pipette tips have a sheath/drip tray        underneath to help capture any drip from the pipette tips after        being used, and also to prevent unwanted contamination of the        instrument.    -   Handle and Flex-Lock allows easy insertion, removal, and        positive location of strip in rack.    -   One or more labels: positioned upward facing to facilitate case        of reading by eye and/or, e.g., a bar-code reader, the one or        more labels containing human and machine readable information        pertaining to the analysis to be performed.

It is to be understood that these dimensions are exemplary. However, itis particularly desirable to ensure that a holder does not exceed thesedimensions so that a rack and an apparatus that accommodates the reagentholder(s) does not become inconveniently large, and can be suitablysituated in a laboratory, e.g., on a bench-top.

Example 2: Disposable Reagent Holder Manufacturing

Simple fixtures can be designed and machined to enable handling andprocessing of multiple strips. There are five steps that can beperformed to produce this component. The disposable reagent holder willbe placed in a fixture and filled with liquids usingmanual/electric-multiple pipetting. Immediately after dispensing allliquids into the strip, foil will be heat sealed to the plastic usingexemplary heat seal equipment (Hix FH-3000-D Flat Head Press) and thefoil trimmed as required. After heat sealing liquids on board, allpellets in tubes can be snapped into the strip, pipette tips can beinserted in their respective sockets, and a barcode label can beaffixed. Desiccant packs can be placed into the blow molded orthermoformed rack designed to house 12 holders. Twelve disposable stripswill be loaded into the rack and then sealed with foil. The sealed begwill be placed into a carton and labeled for shipping.

Example 3: Foil-Sealing of Buffer Containing Reagent Tubes

Tubes containing buffers have to be sealed with high moisture vaporbarrier materials in order to retain the liquid over a long period oftime. Disposable holders may need to have a shelf life of 1-2 years, andas such, they should not lose more than say 10-15% of the liquid volumeover the time period, to maintain required volume of liquid, and tomaintain the concentration of various molecules present in the solution.Moreover, the materials used for construction of the tube as well as thesealing laminate should not react with the liquid buffer. Specialplastic laminates may provide the moisture barrier but they may have tobe very thick (more than 300 μm thick), causing the piercing force to goup tremendously, or of special, expensive polymer (such as Aclar).Aluminum foils, even a thin foil of a few hundred angstrom provides aneffective moisture barrier but hare aluminum reacts with some liquidbuffers, such as sodium hydroxide, even an aluminum foil with a sprayedcoating of a non-reactive polymer may not be able to withstand thecorrosive vapors over a long time. They may react through tiny pin holespresent in the coating and may fill as a barrier over time.

For these reasons, aluminum foils with a laminate structure have beenidentified as a suitable barrier, exemplary properties of which aredescribed below:

1. Sealing

-   -   Heat seals to unitized polypropylene strip (sealing temp        ˜170-180° C.)    -   No wrinkling, cracking and crazing of the foil after sealing

2. Moisture Vapor Transmission Rate (MVTR)

-   -   Loss of less than 10% liquid (20 microliters from a volume of        200 microliter) for a period of 1 year stored at ambient        temperature and pressure. (effective area of transport is ˜63        mm²); Approximate MVTR ˜0.8 cc/m²/day

3. Chemistry

-   -   Ability to not react with 40 mM Sodium Hydroxide (pH<12.6): foil        should have a plastic laminate at least 15 microns thick closer        to the sealed fluid.    -   Ability to not react with other buffers containing mild        detergents

4. Puncture

-   -   Ability to puncture using a p1000 pipette with a force less than        3 lb    -   Before puncturing, a fully supported membrane 8 mm in diameter        will not stretch more than 5 mm in the orthogonal direction    -   After puncturing, the foil should not seal the pipette tip        around the circumference of the pipette.

5. Other Features

-   -   Pin-hole free    -   No bubbles in case of multi-laminate structures.

Example 4: Mechanism of Piercing Through a Plasticized Laminate andWithdrawing Liquid Buffer

The aluminum laminate containing a plastic film described elsewhereherein serves well for not reacting with corrosive reagents such asbuffers containing NaOH, and having the favorable properties ofpierceability and acting as a moisture barrier. However, it presentssome additional difficulties during piercing. The aluminum foil tends toburst into an irregular polygonal pattern bigger than the diameter ofthe pipette, whereas the plastic film lends to wrap around the pipettetip with minimal gap between the pipette and the plastic film. Thediameter of the hole in the plastic film is similar to the maximumdiameter of the pipette that had crossed through the laminate. Thiswrapping of the pipette causes difficulty in dispensing and pipettingoperations unless there is a vent hole allowing pressures to equilibratebetween outside of the tube and the air inside of the tube.

A strategy for successful pipetting of fluid is as follows:

-   -   1. Pierce through the laminate structure and have the pipette go        close to the bottom of the reagent tube so that the hole created        in the laminate is almost as big as the maximum diameter of the        pipette (e.g., ˜6 mm for a p1000 pipette)    -   2. Withdraw the pipette up a short distance so that a small        annular vent hole is left between the pipette and the laminate.        The p1000 pipette has a smallest outer diameter of 1 mm and        maximum outer diameter of 6 mm and the conical section of the        pipette is about 28 mm long. A vent hole thickness of a hundred        microns is enough to create a reliable vent hole. This        corresponds to the pipette inserted to a diameter of 5.8 mm,        leaving an annulus of 0.1 mm around it.    -   3. Withdraw fluid from the tube. Note that the tube is designed        to hold more fluid than is necessary to withdraw from it for a        sample preparation procedure.

Example 5: Foil Piercing and Dissolution of Lyophilized Reagents

The containers of lyophilized reagents provided in conjunction with aholder as described herein are typically sealed by a non-plasticizedaluminum foil (i.e., not a laminate as is used to seal the reagenttubes). Aluminum foil bursts into an irregular polygonal pattern whenpierced through a pipette and leaves an air vent even though the pipetteis moved to the bottom of the tube. In order to save on reagents, it isdesirable to dissolve the reagents and maximize the amount withdrawnfrom the tube. To accomplish this, a star-ridged (stellated) pattern isplaced at the bottom of the container to maximize liquid volumewithdrawn, and flow velocity in between the ridges.

Exemplary steps for dissolving and withdrawing fluid are as follows:

-   -   1. Pierce through the pipette and dispense the fluid away from        the lyophilized material. If the pipette goes below the level of        the lyophilized material, it will go into the pipette and may        cause jamming of the liquid flow out of the pipette.    -   2. Let the lyophilized material dissolve for a few seconds.    -   3. Move pipette down touching the ridged-bottom of the tube    -   4. Perform an adequate number of suck and spit operations (4-10)        to thoroughly mix the reagents with the liquid buffer.    -   5. Withdraw all the reagents and move pipette to dispense it        into the next processing tube.

Example 6: Material and Surface Property of the Lysis Tube

The material, surface properties, surface finish has a profound impacton the sensitivity of the assay performed. In clinical applications,DNA/RNA as low as 50 copies/sample (˜1 ml volume) need to be positivelydetected in a background of billions of other molecules, some of whichstrongly inhibit PCR. In order to achieve these high level ofsensitivities, the surface of the reaction tube as well as the materialof the surface has to be chosen to have minimal binding ofpolynucleotides. During the creation of the injection molding tool tocreate these plastic tubes, the inherent surfaces created by machiningmay have large surface area due to cutting marks as large as tens ofmicrons of peaks and valleys. These surfaces have to be polished to SPIA1/A2 finish (mirror finish) to remove the microscopic surfaceirregularities. Moreover, the presence of these microscopic valleys willtrap magnetic beads (0.5-2μ) at unintended places and cause irregularperformance. In addition to actual surface roughness, the surfacehydrophobicity/surface molecules present may cause polynucleotides tostick at unintended places and reduce sensitivity of the overall test.In addition to the base material uses, such as homogenous polypropyleneand other polymers, specific materials used during the molding of thesetubes, such as mold release compounds or any additives to aid in thefabrication can have a profound impact on the performance of thereactions.

Example 7: Liquid Dispensing Head

Referring to FIGS. 18, 19A-C, and 63, an exemplary liquid dispenser isattached to a gantry, and receives instructions via electrical cable1702. Barcode scanner 1701 is mounted on one face of the liquiddispenser. The gantry is mounted on a horizontal rail 1700 to providemovement in the x-direction. Not shown is an orthogonally disposed railto provide movement in the y-direction. The liquid dispenser comprises acomputer controlled motorized pump 1800 connected to fluid distributionmanifold 1802 with related computer controlled valving 1801 and a 4-uppipetter with individually sprung heads 1803. The fluid distributionmanifold has nine Lec Co. solenoid valves 1801 that control the now ofair through the pipette tips: two valves for each pipette, and anadditional valve to vent the pump. Barcode reader 1701 enables positivedetection of sample tubes, reagent disposables and microfluidiccartridges. The scanner is mounted to the z-axis so that it can bepositioned to read the sample tube, strip, and cartridge barcodes.

Example 8: Integrated Heater/Separator

In FIG. 64 an exemplary integrated magnetic separator and heaterassembly are shown. Magnetic separator 1400 and heater assembly 1401were fabricated comprising twelve heat blocks aligned parallel to oneanother. Each heat block 1403 is made from aluminum, and has an L-shapedconfiguration having a U-shaped inlet for accepting a process chamber1402. Each heat block 1403 is secured and connected by a metal strip1408 and screws 1407. Magnet 1404 is a rectangular block Neodymium (orother permanent rare earth materials, K & J Magnetics, ForcefieldMagnetics) disposed behind each heat block 1403 and mounted on asupporting member. Gears 1406 communicate rotational energy from a motor(not shown) to cause the motorized shaft 1405 to raise and lower magnet1404 relative to each heat block. The motor is computer-controlled tomove the magnet at speeds of 1-20 mm/s. The device further comprises aprinted circuit board (PCB) 1409 configured to cause the heater assemblyto apply heat independently to each process chamber 1402 upon receipt ofappropriate instructions. In the exemplary embodiment, the device alsocomprises a temperature sensor and a power resistor in conjunction witheach heater block.

Example 9: Exemplary Software

Exemplary software accompanying use of the apparatus herein can includetwo broad parts—user interface and device firmware. The user interfacesoftware can allow for aspects of interaction with the user suchas—entering patient/sample information, monitoring test progress, errorwarnings, printing test results, uploading of results to databases andupdating software. The device firmware can be the low level softwarethat actually runs the test. The firmware can have a generic portionthat can be test independent and a portion specific to the test beingperformed. The test specific portion (“protocol”) can specify themicrofluidic operations and their order to accomplish the test.

FIGS. 65A and 65B shows screen captures from the programming interfaceand real time heat sensor and optical detector/monitoring. This realtime device performance monitoring is for testing purposes; not visibleto the user in the final configuration.

User Interface:

A medical grade LCD and touch screen assembly can serve as the userinterface via a graphical user interface providing easy operating andminor troubleshooting instructions. The LCD and touch screen have beenspecified to ensure compatibility of all surfaces with common cleaningagents. A barcode scanner integrated with the analyzer can be configuredto scan the barcode off the cartridge (specifying cartridge type, lot #,expiry date) and if available the patient and user ID from one or moresample tubes.

Example 10: Exemplary Preparatory Apparatus

This product is an instrument that enables 24 clinical samples to beautomatically processed to produce purified nucleic acid (DNA or RNA) inabout half an hour (FIG. 66). Purified nucleic acid may be processed ina separate amplification-detection machine to detect the presence ofcertain target nucleic acids. Samples are processed in a unitizeddisposable strip. preloaded with sample preparation chemistries andfinal purified nucleic acids are dispensed into PCR tubes. Fluidhandling is enabled by a pipetting head moved by a xyz gantry. (FIG. 67)

The System has the following sub-systems:

-   -   Two sample processing racks, each rack processes up to 12        clinical samples in unitized disposable strips    -   Magnetic separator-cum-tube heater assembly (24 heating        stations)    -   A four-probe liquid dispensing head    -   3-axis gantry to move the pipette head    -   Peltier-cooled per-tube holding station to receive the purified        DNA/RNA    -   Control electronics    -   Barcode reader

Operation: The user will got a work list for each sample, whether theywant to extract DNA or RNA for each clinical sample. The sample tubesare placed on the rack and for each sample type (DNA or RNA), the userslides in a unitized reagent disposable (DNA or RNA processing) intocorresponding lane of the rack. The unitized disposable (holder) willhave all the sample prep reagents, process tubes as well as disposablepipettes already prepackaged in it. Once all disposables are loaded intothe rack, the rack is placed in its location on the instrument. Open pertubes are placed in the peltier cooled tube holder where the finalpurified nucleic acid will be dispensed. The user then closes the doorof the instrument and then starts the sample processing using the GUI(Graphical User Interface).

The instrument checks functionality of all subsystems and then reads thebarcode of the sample tubes and the unitized reagent disposable. Anymismatch with a pre-existing work list is determined and errors areflagged, if necessary. The instrument then goes through a series ofliquid processing, heating, magnetic separations to complete the samplepreparation steps for the each of the clinical sample and outputs thepurified nucleic acid into the PCR tube. The basic steps involved ineach sample processing are sample lysis, nucleic acid capture intomagnetic affinity beads, washing of the magnetic beads to removeimpurities, releasing the nucleic acid from the magnetic beads,neutralizing the released DNA and the dispensing into the final PCRtube. These tubes are maintained at 4° C. until all samples areprocessed and user takes away the tube for downstream processing of thenucleic acids.

Example 11: Exemplary Diagnostic Apparatus

The apparatus, in combination with the associated consumables,automatically performs all aspects of nucleic acid testing, includingsample preparation, amplification, and detection for up to 48 samplesper hour with the first 24 results available in less than an hour. Thesystem is easy to use. An operator simply aliquots a portion of thepatient sample into a dedicated tube that contains pre-packaged buffer.The operator places the dedicated tubes into positions on a sample rack.The operator then loads a disposable plastic reagent strip for theappropriate test in the rack. The only other consumable used in theapparatus are microfluidic PCR cartridges for conducting amplificationand detection; each cartridge is capable of performing up to twelve PCRtests and two cartridges can be loaded into the analyzer at once. Shouldthe apparatus require a new PCR cartridge, the analyzer will prompt theoperator to load the cartridge. The analyzer will then prompt theoperator to close the lid to initiate testing. All consumables andsample tubes are barcoded for positive sample identification.

Sample lysis and DNA preparation, which will require approximately halfan hour for a full run of 24 samples, is automatically performed by theanalyzer's robotic and liquid handling components using protocols andreagents located in unitized, disposable plastic strips. The apparatusthen automatically mixes the sample and PCR reagents, and injects themixture into a cartridge that will be automatically processed by anintegrated PCR machine. Rapid, real time PCR and detection requires lessthan 20 minutes. Results, which will be automatically available uponcompletion of PCR, are displayed on the instruments touch screen,printed or sent to the hospital information system, as specified by theuser (or the user's supervisor).

Each instrument can process up to 24 samples at a time with a totalthroughput of 48 samples per hour after the first run. The analyzer isslightly less than 1 m wide and fits easily on a standard lab bench. Alloperations of the unit can be directed using the included barcode wandand touch screen. The analyzer can be interfaced with lab informationsystems, hospital networks, PCs, printers or keyboards through four USBinterfaces and an Ethernet port.

The apparatus has the following characteristics.

Sensitivity: the apparatus will have a limit of detection of ˜50 copiesof DNA or RNA. (and may have a limit of detection as low as 25-30 copiesof DNA/RNA).

Cost per Test: Due to the miniaturized, simplified nature of HandyLabreagents, cartridge and other consumables, the cost of goods per testwill be relatively low and very competitive.

Automation: By contrast with current “automated” NAT systems, which allrequire some degree of reasonably extensive technologist interactionwith the system, through the use of unitized tests and full integrationof sample extraction, preparation, amplification and detection, theapparatus herein will offer a higher level of automation, andcorresponding reduction in technologist time and required skill level,thereby favorably impacting overall labor costs.

Throughput: Throughput is defined as how many tests a system can conductin a given amount of time. The apparatus will be capable of running 45tests per hour, on average.

Time to First Result: In a hospital environment, time to first result isan especially important consideration. The apparatus will produce thefirst 24 results in less than an hour and an additional 24 results everyhalf hour thereafter.

Random Access and STAT: Random access is the ability to run a variety oftests together in a single run and place samples in unassigned locationson the analyzer. Also, with chemistry and immunoassay systems, it isdesirable to be able to add tests after a run has started. This is oftenreferred to as “true random access” since the user is provided completeflexibility with regard to what tests can be run where on an analyzerand when a new sample can be added to a ran. A STAT is a sample thatrequires as rapid a result as possible, and therefore is given priorityin the testing cue on the analyzer. Today, essentially all chemistry andimmunoassay analyzers are true random access and offer STATcapabilities. For NAT, however, very few systems offer any random accessor STAT capabilities. The instrument herein will provide random accessand STAT capabilities.

Menu: The number and type of tests available for the analyzer is a veryimportant factor in choosing systems. The apparatus herein deploys alaunch menu strategy that involves a mix of high volume, “standard”nucleic acid tests combined with novel, high value tests.

The apparatus enables 24 clinical samples to be automatically processedto purify nucleic acid, mix the purified DNA/RNA with PCR reagents andperform real-time PCR in microfluidic cartridge to provide sample toresults in an hour. The exemplary apparatus has two PCR readers, eachcapable of running a 12 lane microfluidic cartridge using an opticalsystem that has dedicated two-color optical detection system. FIG. 68,FIG. 69.

The apparatus has the following sub-systems:

-   -   Two sample processing racks, each rack processes up to 12        clinical samples in unitized disposable strips    -   Magnetic separator-cum-tube heater assembly (24 heating        stations)    -   A four-probe liquid dispensing head    -   3-axis gantry to move the pipette head    -   Two PCR amplification-detection station, each capable of running        a 12-lane microfluidic cartridge and dedicated 2-color optical        detection system for each PCR lane.    -   Control electronics    -   Barcode reader

Pictures of exterior (face on) and interior arm at FIGS. 70, 71,respectively.

Operation: The user will get a work list for each sample, whether theywant to detect certain target analyte (such as GBS, Chlamydia,Gonorrhea, HSV) for each clinical sample. The sample tubes are placed onthe rack and for each sample, the user slides in a unitized reagentdisposable (analyte specific) into corresponding lane of the rack. Theunitized disposable will have all the sample prep reagents, PCRreagents, process tubes as well as disposable pipettes alreadyprepackaged in it. Once all disposables are loaded into the rack, therack is placed in its location on the instrument. The user then placestwo 12-lane microfluidic PCR cartridges in the two trays of the PCRreader. The user then closes the door of the instrument and then startsthe sample processing using the GUI (Graphical User Interface).

The instrument checks functionality of all subsystems and then reads thebarcode of the sample tubes, the unitized reagent disposables and themicrofluidic cartridges. Any mismatch with a pre-existing work list isdetermined and errors are flagged, if necessary. The instrument thangoes through a series of liquid processing, heating, magnetic separationto complete the sample preparation steps for the each of the clinicalsample, mixes the purified nucleic acid with PCR reagents and dispensesthe final mix into a lane of the microfluidic cartridges. After amicrofluidic cartridge is loaded with the final PCR mix, the cartridgetray moves and aligns the cartridge in the reader and the opticaldetection system presses the cartridge against a microfluidic PCR heatersurface. On-chip valves are actuated to close the reaction mix and thenthermocycling is started to initiate the PCR reaction. At each cycle ofPCR (upto 45 cycles), fluorescence from each PCR lane is detected by theoptical detection system (2-colors per PCR lane) and final result isdetermined based on the threshold cycle (Ct).

The sample preparation steps for 24 samples are performed in about 40minutes and the PCR reaction in about 20 minutes.

Sample Reader:

The Reader performs function testing of up to twelve properly preparedpatient samples by PCR process (real-time PCR) when used in conjunctionwith HandyLab microfluidic (test) cartridges. Each unit will employ twoReader Modules for a total of up to twenty four tests. (FIGS. 72A and72B) Operation of the Reader is designed for minimal customerinteraction, requiring the loading and unloading of test cartridgesonly. During the “Load Disposables” sequence, the Reader will present amotor actuated tray for installation of the disposable cartridge.Sliding a small knob located in the front of the tray, a spring loadedprotective cover will raise allowing the test cartridge to be nestedproperly in place. The cover is then lowered until the knob self-locksinto the tray frame, securing the cartridge and preventing movementduring the sample loading sequence.

Once the prepared samples have been dispensed via pipettes into the testcartridge, the tray will retract into the Reader, accurately positioningthe test cartridge beneath the chassis of the optical assembly. Theoptical assembly will then be lowered by a captured screw driven stoppermotor until contact is made with the test cartridge. At this point thetest cartridge is located ⅛″ above the target location on the heaterassembly. As downward motion continues the test cartridge and its holderwithin the tray compress springs on the tray frame (these are used laterto return the cartridge to it's normal position and able to clear theencapsulated wire bonds located on the heater assembly during trayoperation). Movement of the test cartridge and optical assembly iscomplete once contact with the heater assembly is made and a minimum of2 psi is obtained across the two-thirds of the cartridge area about thePCR channels and their controlling gates. At this point the testing ofthe cartridge is performed using the heater assembly, measured withonboard optics, and controlled via software and electronics much in thesame manner as currently operated on similar HandyLab instruments.

Once the functional testing is complete the main motor raises the opticassembly, releasing pressure on the test cartridge to return to it'snormal position. When commanded, the tray motor operating in arack-and-pinion manner, presents the tray to the customer for cartridgeremoval and disposal. When the tray is in the extended position it issuspended above a support block located on the apparatus chassis. Thisblock prevents the cartridge from sliding trough the holder in the trayduring loading and acts as a support while samples are pipetted into thedisposable cartridge. Also provided in this support block is an assistlever to lift and grasp the disposable cartridge during removal. Allcomponents of the tray as well as support block and cartridge liftassist are removable by the customer, without tools, for cleaning andreinstalled easily.

Microfluidic PCR Heater Module.

The microfluidic PCR heater module comprises a glass wafer withphotolithographically defined microheaters and sensors to accuratelyprovide heat for actuation of valves and performing thermocyclingrequired to perform a real-time PCR reaction. The wafer surface hasdedicated individually controlled heating zones for each of the PCRlanes in the microfluidic cartridge. For a 12-up cartridge, there are 12PCR zones and the 24-up cartridge, there are 24 PCR heating zones. Theindividual heaters and sensors are electrically connected to a Printedcircuit board using gold or aluminum wire bonds. A thermally compliantencapsulant provides physical protection the wirebonds. While thepresent device is made on glass wafer, heaters can be fabricated onSi-on-Glass wafers and other polymeric substrates. Each substrate canhave provide specific advantages related to its thermal and mechanicalproperties. Besides using photolithography process, such heatingsubrates can also be assembled using off-the-shelf electronic componentssuch as power resistors, peltiers, transistors, maintaining the upperheating surface of each of the component to be at the same level toprovide heating to a microfluidic cartridge. Temperature calibrationvalues for each temperature sensor may be stored in a EEPROM or othermemory devices co-located in the heater PCBoard.

12-Lane Cartridge:

This 12 channel cartridge is the same basic design that is described inU.S. provisional parent application Ser. No. 60/859,284, filed Nov. 14,2006, with the following modifications: increase the PCR volume from 2μl to 4.5 μl, leading to an increase in the input volume from 4 μl to 6μl. The inlet holes are moved a few millimeters away from the edge ofthe cartridge to allow room for a 2 mm alignment ledge in the cartridge.A similar alignment ledge is also included on the other edge of thecartridge. (FIGS. 31A, 31B)

Enclosure:

The design of the apparatus enclosure must satisfy requirements: forcustomer safety during operation; provide access to power andcommunication interfaces; provide air entry, exit, and filtering;provide one-handed operation to open for installation and removal ofmaterials; incorporate marketable aesthetics.

Cooling:

The cooling for the apparatus will be designed in conjunction with theenclosure and overall system to ensure all assemblies requiring air arewithin the flow path or receive diverted air.

The current concept is for the air inlet to be located on the bottom ofthe lower front panel. The air will then pass through a cleanable filterbefore entering the apparatus. Sheet metal components will direct theair to both the disposable racks and the main power supply. The air willthen be directed through the card cages, around the readers and willexit through slots provided in the top of the enclosure.

Base Plate

The XYZ stage and frame are mounted to the base plate in a way wherethere will be no misalignment between the stage, cartridge and thedisposable. The enclosure is mounted to the base plate. Final design ofthe enclosure determines the bolt hole pattern for mounting. Thebackplane board mounts to the base plate with standoffs. All otherboards mount to the backplane board. The disposable mounts on a rackwhich will be removable from the brackets mounted to the base plate. Thereader brackets bolt to the base plate. Final design of the readerbrackets determines the bolt hole pattern. The power supply mounts tothe base plate. The base plate extends width and lengthwise under theentire instrument.

Example 12: Exemplary High-Efficiency Diagnostic Apparatus

A more highly multiplexed embodiment, also enables 24 clinical samplesto be automatically processed to purify nucleic acids, mix the purifiedDNA/RNA with PCR reagents and perform real-time PCR in a microfluidiccartridge. This product has a single PCR reader, with a scanningread-head, capable of reading up to 4 different colors from each of thePCR lane. The cartridge has 24 PCR channels enabling a single cartridgeto run all 24 clinical samples. In addition, this product has acartridge autoloader, whereby the instrument automatically feeds the PCRreader from a pack of cartridges into the instrument and discard usedcartridge into a waste tray. Diagrams are shown in FIGS. 73, and 74.

The apparatus has the following sub-systems:

-   -   Two sample processing racks, each rack processes up to 12        clinical samples in unitized disposable strips    -   Magnetic separator-cum-tube heater assembly (24 heating        stations)    -   A four-probe liquid dispensing head    -   3-axis gantry to move the pipette head    -   A single PCR amplification-detection station capable of running        a 24-lane microfluidic cartridge and a scanner unit to detect        upto 4 colors from each PCR lane.    -   An autoloader unit to feed 24-lane microfluidic cartridge from a        box into the PCR detection unit.    -   Control electronics    -   Barcode reader

Operation: The user will get a work list for each sample, whether theywant to detect certain target analyte (such as GBS, Chlamydia,Gonorrhea, HSV) for each clinical sample. The sample tubes are placed onthe rack and for each sample, the user slides in a unitized reagentdisposable (analyte specific) into corresponding lane of the rack. Theunitized disposable will have all the sample prep reagents, PCRreagents, process tubes as well as disposable pipettes alreadyprepackaged in it. Once all disposables are loaded into the rack, therack is placed in its location on the instrument. The user then closesthe door of the instrument and then starts the sample processing usingthe GUI (Graphical User Interface).

The instrument checks functionality of all subsystems and then reads thebarcode of the sample tubes, the unitized reagent disposables andpresence of a 24-lane microfluidic cartridge. Any mismatch with apre-existing work list is determined and errors are flagged, ifnecessary. The instrument than goes through a series of liquidprocessing, heating, magnetic separation to complete the samplepreparation steps for the each of the clinical sample, mixes thepurified nucleic acid with PCR reagents and dispenses the final mix intoa lane of a 24-lane microfluidic cartridge. After the microfluidiccartridge is loaded with the final PCR mix, the cartridge is moved andaligned by an automated motorized pusher in the PCR reader. The opticaldetection system, then presses the cartridge against a microfluidic PCRheater surface. On-chip valves are actuated to close the reaction mixand then thermo-cycling is started to initiate the PCR reaction. At eachcycle of PCR (up to 45 cycles), fluorescence from each PCR lane isdetected by the optical detection system (2-color per PCR lane) andfinal result is determined based on the threshold cycle (Ct). The usedcartridge is than pushed out automatically into a waste cartridge bin.

Microfluidic cartridges are stored in a cartridge pack (maximum 24cartridges) and the instrument alerts the user to replace the cartridgepack and empty out the waste cartridge bin once all cartridges from thepeck are used up.

24-Lane Cartridge

The 24-lane cartridge has two rows of 12 PCR lanes. Various views areshown in FIGS. 75-77. The cartridge has 3 layers, a laminate, asubstrate, and a label. The label is shown in two pieces. Each Lane hasa liquid inlet port, that interfaces with a disposable pipette; a 4microliter PCR reaction chamber (1.5 mm wide, 300 microns deep andapproximately 10 mm long), two microvalves on either side of the PCRreactor and outlet vent. Microvalves are normally open and close thechannel on actuation. The outlet holes enables extra liquid (˜1 μl) tobe contained in the fluidic channel incase more than 6 μl of fluid isdispensed into the cartridge.

The inlet holes of the cartridge are made conical in shape and have adiameter of 3-6 mm at the top to ensure pipettes can be easily landed bythe fluid dispensing head within the conical hole. Once the pipettelands within the cone, the conical shape guides the pipette andmechanically scales to provide error free dispensing or withdrawal offluid into the cartridge. The bigger the holes, the better it is toalign with the pipette, however, we need to maximize the number of inletports within the width of the cartridge as well as maintain the pitchbetween holes compatible with the inter-pipette distance. In thisparticular design, the inter-pipette distance is 18 mm and the distancebetween the loading holes in the cartridge is 8 mm. So lanes 1, 7, 11are pipetted into during one dispensing operation; lanes 2, 5, 8 and 12in the next, and so on and so forth.

The height of the conical holes is kept lower than the height of theledges in the cartridge to ensure the cartridges can be stacked on theledges. The ledges on the two long edges of the cartridge enablestacking of the cartridges with minimal surface contact between twostacked cartridges and also help guide the cartridge into the readerfrom cartridge pack (cf. FIGS. 28-33).

Cartridge Autoloader

The Cartridge autoloader consists of a place for positively locking apack of 24 microfluidic cartridges, pre-stacked in a spring-loaded box(e.g., FIG. 33). The box has structural elements on the sides to enableunidirectional positioning and locking of the box in the autoloader(FIG. 33). To load a new box, the user moves a sliding element to theleft of the autoloader, places and pushes the box in the slot andreleases the sliding lock to retain the box in its right location.Springs loaded at the bottom of the box helps push the box up when itneeds to be replaced. The spiral spring present at the bottom of thecartridge pack pushed against the cartridges and is able to continuallypush the cartridge with a force of from 4 to 20 pounds.

The presence or absence of cartridges is detected by reading the barcodeon top of the cartridge, if present.

To start a PCR run, the pipette heed dispenses PCR reaction mix into therequired number of lanes in the top cartridge in the autoloader (e.g.,FIG. 28). The pusher pushes the top cartridge from the autoloader boxinto the two rails that guide the cartridge into the PCR reader. Thecartridge is pushed to the calibrated location under the reader and thenthe optics block is moved down using a stepper motor to push thecartridge against the microheater surface. The bottom of the opticsblock (aperture plate) has projections on the sides to enable thecartridge to be accurately aligned against the apertures. The steppermotor pushes the cartridge to a pre-calibrated position (e.g., FIG. 30)which provides a minimum contact pressure of 1 psi on the heatingsurface of the microfluidic cartridge.

After the PCR reaction is complete, the stepper motor moves up 5-10 mmaway from the cartridge, relieves the contact pressure and enables tocartridge to travel in its guide rails. The pusher is activated and itpushes the cartridge out to the cartridge waste bin (e.g., FIG. 32).After this step, the pusher travels back to its home position. Duringits beck travel, the pusher is able to rise above the top of thecartridge in the cartridge pack because it has a angular degree offreedom (sec figure). A torsion spring ensures the pusher comes back toa horizontal position to enable it to push against the next cartridge inqueue. The pusher is mechanically attached to a timing belt. The timingbelt can be moved in either direction by turning a geared motor. Thepusher is mounted to a slider arrangement to constrain it to move inonly one axis (see, e.g., FIG. 31).

The cartridge pushing mechanism can also be made to not only push thecartridge from the autoloader box to the detection position, but also beused to move it back to the autoloading position. This will enableunused lanes in the microfluidic cartridge to be used in the next PCRrun.

The cartridge autoloading box is also designed so that once all thecartridges are used, the box can be easily recycled or new cartridgesadded to it. This reduces the cost to the customer and the manufacturer.

Reader

The reader consists of an optical detection unit that can be pressedagainst a 24-lane microfluidic cartridge to optically interface with thePCR lanes as well as press the cartridge against a microfluidic heatersubstrate (FIG. 78). The bottom of the optics block has 24 apertures(two rows of 12 apertures) that is similar in dimension of the PCRreactors closest to the cartridge. The aperture plate is made of lowfluorescent material, such as anodized black aluminum and duringoperation, minimized the total background fluorescence while maximizingthe collection of fluorescent only from the PCR reactor (FIGS. 79A and798). The bottom of the aperture plate has two beveled edges that helpalign two edges of the cartridges appropriately such that the aperturesline up with the PCR reactors. (FIGS. 80, 81)

The optical detection units (total of 8 detection units) are assembledand mounted onto a sliding rail inside the optical box so that theoptical units can be scanned over the apertures (FIG. 82). Each unit isable to excite and focus a certain wavelength of light onto the PCRreactor and collect emitted fluorescence of particular wavelength into aphotodetector. By using 4 different colors on the top 4 channels andrepeating the 4 colors in the bottom channels, the entire scanner canscan up to 4 colon from each of the PCR lanes.

The optics block can be machined out of aluminum and anodized orinjection molded using low fluorescence black plastic (FIG. 83).Injection molding can dramatically reduce the cost per unit and alsomake the assembly of optics easier. The designed units can be stackedback-to-back.

Example 13: Exemplary Electronics for Use with Preparatory andDiagnostic Apparatuses as Described Herein

There are multiple independent software modules running on dedicatedhardware: Described herein are exemplary specifications for theelectronics used in the diagnostic (PCR) system. Additional informationrelated to the PCR System is described elsewhere herein. In someembodiments, the PCR system includes eighteen printed circuit boards(PCBs) of nine different types. Referring to FIG. 86, the system cancontain three multiplex (MUX) boards 100 a-c, two of which (micro-heaterMUX boards 100 a-b), can each be used to run a micro-heater board 110a-b and the third (lysis heater MUX board 100 c) can run one or morelysis heater boards 116 and 117. Each of the three MUX boards 100 a-ccan be controlled by a PC processor board via an Ethernet port. The twomicro-heater boards 110 a-b, each controlled by one of the MUX boards100 a-b, heat micro-zones on the microfluidic cartridge. In someembodiments, the system includes the two lysis heater boards 116 and117, controlled by the lysis heater MUX board 100 c, that heat lysistubes in each of the two 12 sample racks.

Still referring to the PCBs included in the PCR system, the system caninclude two 12-channel optical detection boards 130 a-b that can eachdetect optical fluorescence emitted by microfluidic cartridge chemistry.The optical detection boards can be controlled by one or more of the MUXboards 100 a-c, using SPI, over a RS-422 interface. The system caninclude three motor control boards 140 a-c, where one board (e.g., motorcontrol board 140 c) can control two magnetic separation motors (notshown), and the remaining two motor control boards (e.g., motor controlboards 140 a-b) can each run one reader tray motor (not shown) and onereader pressure motor (not shown). The motor control board running themagnetic separation motors (e.g., motor control board 140 c) can becontrolled via RS-485 interface from the lysis heater MUX board 100 cand the two motor control boards 140 a-6, each running one reader traymotor and one reader pressure motor, can be controlled via RS-485interface by the micro-heater MUX boards 100 a-b. The system can alsoinclude one PC processor board 150, which directs the overall sequencingof the system and can be controlled via external Ethernet and USBinterfaces, and one PC processor base board 160, which provides internalinterfaces for the PC processor board 150 to the remainder of the systemand external interfaces. The system can include one main backplane 180that interconnects all system boards, one motor control backplane 190that interconnects the motor control boards 140 a-c to the mainbackplane 180 and gantry (not shown), and two door sensor boards (notshown). One door sensor board provides an interconnect between the frontdoor solenoid locks and the PC processor base board 160 and the otherdoor sensor board provides an interconnect between the position sensorsand the PC processor base board 160.

In some embodiments, the PCR system can include the off-the-shelf PCprocessor board 150. The PC processor board 150 can be an ETX formfactor board that includes one 10/100 BASE-T Ethernet port, four USBports, one analog VGA display port, two UART ports, one real-time clock,one parallel port, one PS2 keyboard port, one PS2 mouse port, stereoaudio output, one IDE interface, and one I2C interface.

Referring to FIG. 87, the system can also include the PC processor baseboard 160 that includes a five port 10/100 BASE-T Ethernet bridge 161for internal communication, one of which can be connected to the 10/100BASE-T Ethernet port of the PC Processor board 150, another of which canbe for diagnostic use (with a connector inside system cover), and threeof which can communicate with the three MUX boards 100 a-c (one port foreach MUX board 100 a-c) through the backplane 180. The PC processor baseboard 160 can also include one USB to 10/100 BASE-T Ethernet port 162for external Ethernet connections, one four port USB hub 163 forexternal connections, one external VGA connector 164, one internal PS2Mouse connector 165 (with a connector inside the system cover), and oneinternal PS2 Keyboard connector 166 (with a connector inside the systemcover. The PC processor base board 160 can also include one internalstereo audio output 167 to on board speakers 168, one internalCompactFlash connector 169 from an IDE port (with a connector inside thesystem cover), and one internal RS-232 interface 170 from a UART port(with a connector inside the system cover). Additional componentsincluded in the PC processor base board can include one internal RS-485interface 171 from a UART port (with a connector inside the systemcover), one internal temperature sensor 172 connected to the I2Cinterface, a battery for the real-time clock, and one parallel port 173.The parallel port 173, with connectors inside the system cover, can beinternally connected as follows: one bit can be used to drive a highcurrent low side switch for the two door solenoids, one bit can be usedto generate a processor interrupt when either door sensor indicates thata door is opened, three bits can be used to program the EEPROM forconfiguring the Ethernet bridge 161, and two bits can be connected tothe Ethernet bridge management interface (not shown). The remaining bitscan remain unassigned, with optional pull-up and pull-down resistors,and be brought out to a 10 pin Phoenix contact header.

Referring now to FIG. 88, in some embodiments, the system can includethe three MUX boards 100 a-c. While FIG. 88 depicts exemplary MUX board100, each of the three MUX boards 100 a-c can include one or more of thefeatures described below. The MUX board 100 a can include 96 pulse widthmodulated (PWM) controlled heating channels with heaters (about 33 ohmto about 150 ohm) heaters, that can support 20 or 24 volt (voltageexternally provided) drives with a maximum current of about 800 mA. EachPWMs can be 12-bit with programmable start and stop points, can have 1microsecond resolution, and can have a maximum duty cycle of about 75%.Each PWM period is programmable and is preferably set to 4 ms. The MUXboards can include a 4-wire RTD/heater connection with precision 1 mAsense current that can accommodate about 150 ohm to about 2500 ohmresistive temperature devices and have a measurement accuracy of +/=0.5ohms. The thermal measurement sample period of the MUX boards is 32 msincluding 8×PWM periods where 12 16-bit ADCs 101 a sample 8 successivechannels each. The MUX address can be tagged to the ADC data.

Still referring to the MUX board 100 a depicted in FIG. 88, an RS-422optics board interface 102 a that interconnects over the backplane 180and transfers data over a 4 wire SPI interface using local handshakesignals and interrupts can be included on the MUX board 100 a. The MUXboard 100 a can also include a 10/100 BASE-T Ethernet interface 103 athat interconnects to the system over the backplane 180 and an RS-485interface 104 a that interconnects to the motor controller 140 a overthe backplane 180.

Referring now to FIG. 89, in some embodiments, the system can includethe optical detection boards 130 a-b. While FIG. 89 depicts exemplaryoptical detection board 130 a, each of the optical detection boards 130a-b can include one or more of the features described below. The opticaldetection board 130 a can include a 12-channel optics board designmodified to use an RS-422 interface 131 a. The optical detection board130, can include 12-3 Watt, blue LEDs 132 a driven with about 6 V atabout a 625 mA maximum. An exemplary LED used in the detection board 130a is the Luxeon K2 emitter producing blue light at a wavelength of about470 nm using about 27 mW @ 700 mA. The optical detection board 130 a canalso include 12-3 Watt, amber LEDs 133 a driven with about 6 V at abouta 625 mA maximum. An exemplary LED used in the detection board 130 a isthe Luxeon K2 emitter producing amber light at a wavelength of about 590nm using about 60 mW @ 700 mA. The detection board 130 a can include 24lensed silicon photodiode detectors 134 a, an example of which is theHamamatsu S2386-18L. These photodiode detectors 134 a are designed in acommon TO-18 package. The detection board 130 a can also include anMSP430 processor 135 a with two PWM channels, one for the blue channeland one for the amber channel. The board 130, can include individual LEDenables 136 a and 137 a for each of the 12 color pairs set over thelocal SPI bus.

The PCR system can include a lysis heater board that provides andmonitors heating to the lysis tubes. The heater board can include 12-70Watt TO-247 power resistors (provide heat to the lysis tubes) designedto be fed 24V from one or more of the MUX boards 100 s-c (e.g., MUXboard 100 c) and 12-2000 ohm Resistive Temperature Devices (RTD) tomonitor the temperature of the lysis tubes. Optional resistors can beincluded to modify the full scale range of the RTDs. Included on thelysis heater board is a serial EEPROM that may hold a board serialnumber and can be used to identify the board type and revision level tosoftware.

Referring now to FIG. 90, in some embodiments, the system can includethe micro-heater boards 110 a-b. While FIG. 90 depicts exemplarymicro-heater board 110 a, each of the micro-heater boards 110 a-b caninclude one or more of the features described below. In someembodiments, the system can include the micro-heater board 110, thatincludes a serial EEPROM and two optical interrupters. The serial EEPROMmay hold a board serial number, can hold RTD calibration data, and canbe used to identify the board type and revision level to software. Theoptical interrupters can be used to sense the reader tray position forthe motor control board 140, and sends the information to the Blue Cobra(motor controllers), which processes the information on the positions ofthe reader trays and accordingly controls the power to the emitterssupplied by the motor control board 140 a. The micro-heater board 110 acan provide connections to the 96 channel micro-heater plate and controlthe 96 multiplexed heater/RTD devices to control cartridge featuretemperature. The heater/RTD devices can be between about 50 ohms toabout 500 ohms. The micro-heater board 110 a can bridge the RS-422interface from, for example, the MUX board 100 a to the opticaldetection board 130 a. The connection from the micro-heater board 110,to the MUX board 100, is over the backplane 180, while the connection tothe optics board 130, is over a 40 pin FFC cable.

Referring now to FIG. 91, in some embodiments, the system can includethe motor control boards 140 a-c. While FIG. 91 depicts exemplary motorcontrol board 140 a, each of the motor control boards 140 a-c caninclude one or more of the features described below. In someembodiments, the system can include the motor control board 140, thatcan control two micro-stepping motors 141 a and can be connected to thebackplane 180 via a RS-485 interface. The output to the motors can be upto 24 V supplied externally through the backplane 180. The outputcurrent can be jumper selectable. Exemplary output currents that can beselected via jumper settings can include about 700 mA, about 1.0 A, or2.3 A. The motor control board 140 a includes open collector TTLinterrupt output to the MUX board 100 a and flag inputs. The flag inputscan provide 1.5 V power output to the sensors and can be switched on andoff by software.

Limit switches are placed on the extreme locations of each axis, e.g.,x-minimum and x-maximum, that turns off the power to the motor drivingthat axis incase of a malfunction happens and the pipette head moves outof the designed working distance. Optional pull-up and pull-down areused with the output of the optical interrupters.)

In some embodiments, the system can include one or more interconnectionboards, such as the main backplane 180. The main backplane 180 caninterconnect other PCBs, such as the MUX boards 100 a-c., PC processorbase board 160, and heater interconnect boards. The main backplane 180can cable to the motor control backplane 190 and to two lysis heaterboards. The main backplane 180 can distribute power and signaling,implement 10/100 BASE-T Ethernet and RS-485 over the backplane 180, andsupplies voltages from an external connector. Exemplary voltagessupplied include +3.3 V, +5.0 V, +12.0 V, −12.0 V, +20.0 V, and +24.0 V.

The system can include the motor control backplane 190 that candistribute power and signaling for all of the motor control boards 140a-c. The motor control backplane 190 can supply +5.0 V and 24.0 V froman external connector. The motor control backplane 190 can include 1slot for the RS-485 signaling from each of the two MUX boards 100 a-b(total of 2 slots), 6 slots for the RS-485 signaling from the lysisheater controlling MUX board 100 c, and one connector that providesRS-485 signaling and power to the gantry. The motor control backplane190 can provide pull-up and pull-down resistors to handle floatingbuses.

In some embodiments, the system can include a heater interconnect boardand a door sensor board. The heater interconnect board can connect themicro-hater boards 110 a-b to the main backplane 180 using a physicalinterconnect only (e.g., no active circuits). The door sensor board canprovide a cable interface and mixing logic from the opticalinterrupters, which sense the door is open, and provide a mounting andcabling interface to the door lock solenoid.

Example 14: Exemplary Software for Use with Preparatory and DiagnosticApparatuses as Described Herein

There are multiple independent software modules running on dedicatedhardware:

-   -   Reader (2);    -   Sample-Prep (1);    -   User Interface (1);    -   Detector(2);    -   Motor control (8)

Inter-module communication among is via an internal Ethernet bus,communication with the user interface is via a high speed SPI bus andcommunication with motor control via a RS485 serial bus.

The Reader and Sample-Prep software run on identical hardware and are assuch identical incorporating the following functions:

-   -   Script Engine (a parameterized form of a protocol)    -   Protocol Engine    -   Temperature Control (Microfluidics, lysis, release)    -   Motor control (via external motor control modules). Salient        features of the motor control software are:    -   Command/reply in ASCII and addressing capability to allow daisy        chaining of communication link.    -   Detection (via external detector modules) Detector module        controls the LED illumination and photo detector digitization.

The user interface is implemented as a program running under Linuxoperating system on an embedded x86 compatible PC. The followingfunctions are addressed:

Graphical User Interface

-   -   Test control and monitor    -   Test result storage and retrieval Network connectivity via        Ethernet (to lab information systems)    -   USB interface    -   Printer    -   Scanner (Internal and external)    -   Keyboard    -   Mouse    -   Door lock and sense

Example 15: Exemplary Chemistry and Processes of Use Chemistry Overview:

The chemistry process centers around the detection and identification oforganisms in a clinical specimen, by virtue of detecting nucleic acidsfrom the organism in question. This involves isolation of nucleic acidsfrom target organisms that are contained in a clinical specimen,followed by a process that will detect the presence of specific nucleicacid sequences. In addition to target detection, an internal positivecontrol nucleic acid will be added to the collection buffer, and will betaken through the entire extraction and detection process along withtarget nucleic acids. This control will monitor the effectiveness of theentire process and will minimize the risk of having false negativeresults.

Nucleic Acid Extraction and Purification:

Nucleic acid extraction procedures begin with the addition of a clinicalspecimen to a prepared specimen collection solution. This can be doneeither at a specimen collection site, or at the testing site. Twocollection solution formats will be available: one for body fluids, andone for swab specimens. Collection solutions used at collection siteswill serve as specimen transport solutions, and therefore, this solutionmust maintain specimen and analyte integrity.

The extraction and purification procedure, which is entirely automated,proceeds as follows:

Target organisms are lysed by heating the detergent-containingcollection solution.

Magnetic beads, added to the specimen/collection solution mix,non-specifically bind all DNA that is released into the solution.

Magnetic beads are isolated and are washed to eliminate contaminants DNAis released from the beads using high pH and heat.

DNA containing solution is removed and neutralized with a buffer

Nucleic Aid Amplification:

Nucleic acids that have been captured by magnetic beads, washed,released in high pH, and neutralized with buffer, are added to a mixtureof buffers, salts, and enzymes that have been lyophilized in a tube. Themixture is rapidly rehydrated, and then a portion of the solution isloaded onto a microfluidic cartridge. The cartridge is then loaded intothe amplification instrument module, which consists of a heating unitcapable of thermal cycling, and an optical detection system. Detectionof target nucleic acids proceeds as follows:

-   -   The liquid in scaled in a reaction chamber.    -   Rapid thermal cycling is used to potentiate the Polymerase Chain        Reaction (PCR), which is used to amplify specific target DNA.    -   Amplified DNA fluoresces, and can be detected by optical        sensors.    -   A fluorescent probe “tail” is incorporated into each amplified        piece of DNA    -   At a specific temperature, the probe adopts a conformation that        produces fluorescence (this is termed a “scorpion” reaction, see        FIG. 84).    -   Fluorescence is detected and monitored throughout the reaction.

Extraction and Amplification/Detection Process:

Extensive bench-scale testing has been performed to optimize the nucleicacid extraction chemistry, including the collection buffer, the washbuffer formulation, the release solution formulation, and the PCRreagent mixes. The fully automated method of extraction, followed by12-up PCR, was able to provide very high sensitivity consistently at 150copies/sample.

Examples: Chlamydia in Urine (50150); Gonorrhoea in Urine; GBS inPlasma.

Various detection chemistries such as Taqman, Scorpion, SYBRg Green workreliably in the microfluidic cartridge.

Reagent Manufacturing

Feasibility studies were conducted in order to determine whether PCRreagents could be lyophilized in PCR tubes besides the use of 2 μllyophilized pellets. The studies have indicated that sensitivity ofreactions performed using tube-lyophilized reagents is equivalent tothat of wet reagents or 2 μl pellet reagents, so feasibility has beenproven. Stability studies for this format indicate similar stabilitydata. We have seen 2 microliter lyophilized PCR pellets to be stable toup to 2 years at room temperature, once sealed in nitrogen atmosphere.

Manufacturing Overview: Manufacturing the components of the system canbe accomplished at HandyLab, Inc., Ann Arbor, Mt. The manufacturing taskhas been split into five areas that consist of: chemistry manufacture,disposable strip, collection kit, cartridge and analyzer.

Chemistry Manufacturing: There are currently seven individual, blendedchemistry components identified for potential use with the systemdescribed herein. Mixing, blending and processing reagents/chemicals canbe performed at HandyLab, Inc., with existing equipment already inplace. Additional tooling and fixtures will be necessary as the productmatures and we ramp to high volume production, but initial costs will beminimal.

Collection buffer, wash, release & neutralization liquids are simplerecipes with very low risk, and can be made in large batches to keeplabor costs of mixing/blending at or below targeted projections. Theywill be mixed and placed into intermediate containers for stock, andthen issued to Disposable Strip Manufacturing for dispensing. MatureSOP's are in place from prior project activity.

Affinity Beads (AB) have good potential to be stored and used as aliquid in the strip, but design contingencies for using a lyophilizedpellet are in place as a back up. It is critical to keep the beadssuspended in solution during dispense. Dispense equipment (e.g.,manufactured by Innovadyne) that provides agitation for continuoussuspension during dispense has been identified for purchase oncestability has been proven for liquid AB storage in the strip. Theprocess to manufacture and magnetize the Affinity Beads spans a 9 hourcycle time to produce a batch of 2,000 aliquots, but that same timeperiod can be used for scaled up recipe batches once we ramp into highvolume production. This item has the highest labor content of allchemistry manufacture that is currently required for the apparatus.

PCR reagents/enzymes will be freeze-dried in our existing lyophilizingchamber (Vitlis Genesis) but will not require spherical pelletformation. Instead, the mixture is being dispensed into, and thenlyophilized, inside the end-use tube. First the chemistries are mixedper established SOPs, and then the following steps are performed toaccomplish lyophilization: Individual tubes are placed into arack/fixture, and the solution is dispensed into each, using existingequipment (EFD Ultra Dispense Station.). The filled rack will be placedinside a stainless steel airtight box (modified to accept stoppers inthe lid) and then placed into the lyophilization chamber and the dryingcycle commences unattended. During lyophilization, the stoppers are in araised position allowing air/nitrogen to circulate into, and moisture toexit the stainless box holding racks of vials. At the end of the cycle,the shelves of our lyophilition chamber lower to seat the stoppers intothe lid, forming a seal while still inside the closed chamber, in amoisture free nitrogen atmosphere. The steel boxes are then removed fromthe chamber, and each rack inside shall be processed in a singleoperation to seal all vials in that rack. Immediately after sealing, thevials will be die cut from the foil in one operation, allowingindividual vials to be forwarded to the Disposable Manufacturing areafor placement into a strip. Internal Control will either be added to anexisting solution, or will be dispensed into its own cavity in themanner of the collection buffer, wash, neutralization, and releasesolutions. If lyophilization is required, it will be accomplished in thesame manner as the PCR chemistry, and later snapped into the strip.Shelf life stability studies are underway.

Collection Kit Manufacturing

The collection kit will be processed manually in house for initialquantities. Initial quantities will not require capital expenditures aswe have all equipment necessary to enable us to meet projections through2008. We will be using our existing equipment (EFD 754-SS Aseptic Valve& Valvemate 7000 Digital Controller) to fill the collection vial. Thevials have a twist-on top that will be torqued, and the vial will have aproprietary ID barcode on each vial. 24 vials will be placed into areclosable plastic bag and placed into a carton for shipping.

Place vials into rack.

Dispense solution into vials.

Install and torque caps.

Label vials.

Bag vials and label bag.

Place vial beg and instructions/insert into carton, close and label.

Cartridge Manufacturing:

Existing semi-automatic equipment for laminating & waxing (Think &Tinker DF-4200, & Asymtek Axiom Heated Jet Platform, respectively) willbe utilized to meet all cartridge manufacture requirements. Thefootprint of the 12-up disposable is the same as the RTa10 cartridge, soadditional fixtures are not necessary.

Laminate micro substrate & trim excess.

Fill valves with hot wax & inspect

Apply label & barcode.

Band 24 pieces together.

Bag & seal banded cartridges, label bag.

Place bag & insert(s) into carton, seal and label.

This portion of the product is relatively simple, although there is adifference between the automated (as used herein) and the stand-alone12-up cartridge. Venting will not be required on the cartridge, whicheliminates the most time consuming process for cartridge manufacture,along with the highest risk and highest cost for fully integratedautomation. Over 1,000 pieces or the 12-up with venting have beensuccessfully produced.

Example 16: Exemplary Chemistry Processes Sample Pre-Processing

For Urine Sample: Take 0.5 ml of urine and mix it with 0.5 ml ofHandyLab collection buffer. Filter the sample through HandyLab Inc.'spre-filter (contains two membranes of 10 micron and 3 micron pore size).Place the sample tube in the position specified for the external sampletube in the 12-up rack.

For Plasma Sample: Take 0.5 ml of plasma and mix it with 0.5 ml ofHandyLab collection buffer. Place the sample tube in the positionspecified for the external sample tube in the 12-up rack.

For GBS swab samples: Take the swab sample and dip it in 1 ml ofHandyLab collection buffer. Place the sample tube in the positionspecified for the external sample tube in the 12-up rack.

The HandyLab sample collection buffer contains 50 mM Tris pH 7, 1%Triton X-100, 20 mM Citrate, 20 mM Borate, 100 mM EDTA, plus 1000 copiesof positive control DNA.

Loading the instrument and starling sample processing

-   -   1. Load PCR tube containing PCR master mix in one of the        specified snap-in location of the unitized disposable.    -   2. Load PCR tube containing PCR probes and primers for the        target analyte under consideration in the specified location of        the unitized disposable.    -   3. In case of two analyte test, load PCR tube containing probes        and primers for second analyte in the specified location of the        unitized disposable.    -   4. Load the unitized disposable in the 12-up rack in the same        lane as the sample tube under consideration.    -   5. Prepare and load unitized reagent strips for other samples in        consideration.    -   6. Load the 12-up rack in one of the locations in the        instrument.    -   7. Load 12-up cartridge in the cartridge tray loading position.    -   8. Start operation.        Liquid processing steps    -   1. Using Pipette tip #1, the robot transfers the clinical sample        from the external sample tube to the lysis tube of the unitized        disposable strip.    -   2. Using the same pipette tip, the robot takes about 100 μl of        sample, mixes the lyophilized enzyme and affinity beads,        transfers the reagents to the lysis tube. Mixing is performed in        the lysis tube by 5 suck and dispense operations.    -   3. The robot places pipette tip #1 at its designated location in        the unitized disposable strip.    -   4. Heat the lysis tube to 60 C and maintain it for 10 minutes.    -   5. After 5 minute of lysis, the robot picks up pipette tip #1        and mixes the contents by 3 suck and dispense operations.    -   6. The robot places pipette tip #1 at its designated location in        the unitized disposable strip.    -   7. After 10 minutes of lysis, a magnet is moved up the side of        the lysis tube to a middle height of the sample and held at that        position for a minute to capture all the magnetic beads against        the wall the tube.    -   8. The magnet is brought down slowly to slide the captured beads        close to the bottom (but not the bottom) of the tube.    -   9. Using pipette tip #2, aspirate all the liquid and dump it        into the waste tube.    -   10. Aspirate a second time to remove as much liquid as possible        from the lysis tube.    -   11. Using the same pipette tip #2, withdraw 100 μl of wash        buffer and dispense it in the lysis tube. During this dispense,        the magnet is moved downwards, away from the lysis tube.    -   12. Perform 15 mix steps to thoroughly mix the magnetic beads        with the wash buffer.    -   13. Wait for 30 seconds.    -   14. Move magnet up to capture the beads to the side and hold for        15 seconds.    -   15. Using pipette tip #2, aspirate wash buffer twice to remove        as much liquid as possible and dump it back in the wash tube.    -   16. Move magnet down away from the lysis tube.    -   17. Place pipette tip #2 in its specified location of the        unitized disposable strip.    -   18. Pick up a new pipette tip (tip #3) and withdraw 8-10 μl of        release buffer and dispense it over the beads in the lysis tube.    -   19. Wait for 1 minute and then perform 45 mixes.    -   20. Heat the release solution to 85° C. and maintain temperature        for 5 minutes.    -   21. Place pipette tip #3 in its specified location of the        unitized disposable strip.    -   22. Bring magnet up the tube, capture all the beads against the        tube wall and move it up and away from the bottom of the tube.    -   23. Pick up a new pipette tip (tip #4) and withdraw all the        release buffer from the lysis tube and then withdraw 3-10 μl of        neutralization buffer, mix it in the pipette tip and dispense it        in the PCR tube. (In case of two analyte detections, dispense        half of the neutralized DNA solution into first PCR tube and the        rest of the solution in the second PCR tube.    -   24. Using pipette tip #4, mix the neutralized DNA with the        lyophilized reagents by 4-5 suck and dispense operations and        withdraw the entire solution in the pipette tip.    -   25. Using pipette tip #4, load 6 μl of the final PCR solution in        a lane of the 12-up cartridge.

The usage of pipette heads during various processes is shownschematically in FIGS. 85A-C.

Real-Time PCR

After all the appropriate PCR lanes of the PCR cartridge is loaded withfinal PCR solution, the tray containing the cartridge moves it in thePCR Analyzer. The Cartridge is pressed by the Optical detectionread-head against the PCR heater. Heaters activate valves to closeeither ends of the PCR reactor and real-time thermocycling processstarts. After completing appropriate PCR cycles (˜45 cycles), theanalyzer make a call whether the sample has the target DNA based on theoutput fluorescence data.

Pipette Detection

The pipette bead has 4 infrared sensors for detecting the presence ofpipettes. This is essential to ensure the computer positively knows thata pipette is present or missing. Since pipettes are picked up usingmechanical forcing against the pipette and also dispensed usingmechanical motion of a stripper plate, pipette sensing helps preventingerrors that otherwise may happen.

Force Sensing of the Pipette Head

The multi-pipette head is assembled in such a way and a force sensorinterfaced with it so that any time the pipette head sets against thedisposable pipette(s) or the picked pipettes are forced through thelaminate in the reagent disposable or the pipette is forced against thebottom of the tubes in the reagent disposable, an upward force acts onthe pipette head through the pipette holding nozzle or the pipettesitself. The entire head is pivoted, as shown in Figure and any foreacting on the head causes a set-screw on the upper part of the head topress against a force sensor. This force sensor is calibrated forvertical displacement of the head against a non-moving surface. Usingthis calibration, it can be determined when to stop moving the head inthe z-direction to detect whether pipettes are properly seated or ifpipettes hit tube bottoms.

Alignment of Pipette Tips while Loading PCR Reagents into theMicrofluidic Cartridge

The pipettes used in the apparatus can have volumes as small as 10 μl toas large as 1 ml. Larger volume pipettes can be as long as 95 umm (p1000pipette). When 4 long pipette tips are sprung from the head, even a 1″misalignment during seating can cause the tip to be off-center by 1.7mm. As it is impossible to have perfect alignment of the tip both at thetop where it is interfaced with the tip holder and the bottom, itbecomes necessary to mechanically constrain all the tips at anotherlocation closer to the bottom. We have used the stripper plate, having adefined hole structure to use it to align all the tips. The stripperplate hole clears all the 4 pipette tips when they are picked up. Afterthe tips are properly seated, the stripper plate is moved in the x-axisusing a motor to move all the pipette against the notch provided in thestripper plate (see FIG. 46b ). Now all the pipettes land on thecartridge inlet holes with ease.

Sample Preparation Extension

The current technology describes details of processing clinical samplesto extract polynucleotides (DNA/RNA). The same product platform can beextended to process samples to extract proteins and other macromoleculesby changing the affinity molecules present in the magnetic beads. Theamplification-detection platform can also be used to perform otherenzymatic reactions, such as immunoPCR, Revne-Stranscript as PCR, TMA,SDA, NASBA, LAMP. LCR, sequencing reactions etc. The ample preparationcan also be used to prepare samples for highly multiplexed microarraydetection as well.

Example 16: Exemplary Material for RNA-Affinity Matrix

An exemplary polynucleotide capture material preferentially retainspolynucleotides such as RNA on its surface when placed in contact with aliquid medium that contains polynucleotides mixed with other speciessuch as proteins and peptides that might inhibit subsequent detection oramplification of the polynucleotides.

The exemplary polynucleotide capture material is: Polyamidoamine (PAMAM)Generation 0, available from the Sigma-Aldrich Chemical Company(“Sigma-Aldrich”), product number 412368 PAMAM is a dendrimer whosemolecules contain a mixture of primary and tertiary amine groups. PAMAM(Generation 0) has the structure shown herein.

The PAMAM, during use, is immobilized on a solid support such acarboxylated beads, or magnetic beads. The polynucleotide capturematerial comprises polycationic molecules during an operation ofpolynucleotide capture. Affinity between the material andpolynucleotides is high because polynucleotides such as DNA and RNAtypically comprise polyanions in solution.

After polynucleotide molecules are captured on a surface of thematerial, and remaining inhibitors and other compounds in solution havebeen flushed away with an alkaline buffer solution, such as aqueous 0.1mM Tris (pH 8.0), the polynucleotides may themselves be released fromthe surface of the material by, for example, washing the material with asecond, more alkaline, buffer, such as Tris having a pH of 9.0.

Exemplary protocols for using PAMAM in nucleic acid testing are found inU.S. patent application Ser. No. 12/172,214 filed Jul. 11, 2008,incorporated herein by reference.

Example 17: Exemplary Material for DNA-Affinity Matrix

The exemplary polynucleotide capture material is: Polyethyleneimine(PEI), available from the Sigma-Aldrich Chemical Company(“Sigma-Aldrich”), product number 408719.

Exemplary protocols for using PEI in nucleic acid testing are found inU.S. paten application Ser. No. 12/172,208 filed Jul. 11, 2008,incorporated herein by reference.

Example 18: Exemplary Apparatus

Described herein are exemplary specifications for the mechanical designof the PCR system. In some embodiments, the system can be about 28.5inches deep, or less, and about 43 inches wide, or less, and weightabout 250 pounds or less. The system can be designed with a useful lifeof about 5 years (e.g., assuming 16,000 tests per year) and can bedesigned such that the sound level for this instrument (duringoperation) does not exceed 50 dB as measured 12 inches from theinstrument in all ordinate directions. In some embodiments, the exteriorof the system can be white with texture.

Referring to the overall system, in some embodiments, criticalcomponents of the system can remain orthogonal or parallel (asappropriate) to within 0.04 degrees. Exemplary critical components caninclude motion rails, pipettes, nozzles (e.g., axially as individualnozzles, linearly as an array of four nozzle centroids, or the like),lysis heaters, major edges of the installed cartridge holder in thereader drawer, the hut face of the separation magnets, and the like. Inthe following descriptions, the X-axis (or X direction) refers to theaxis extending from left to right when facing the front of the system,the Y-axis (or Y direction) refers to the axis extending from back tofront when facing the front of the system, and the Z-axis (or Zdirection) refers to the axis extending up from the bottom when facingthe front of the system. As viewed from the top of the instrument, thecentroid of the leftmost pipette nozzle on the Z-payload (as viewed fromthe front of the instrument) can be capable of unobstructed travel inthe X direction from a point 80 mm from the outermost left baseplateedge to a point 608 mm from the outermost left baseplate edge and can becapable of unobstructed travel in the Y direction from a point 60 mmfrom the outermost front baseplate edge to a point 410 mm from theoutermost front baseplate edge.

Still referring to the system, as viewed from the front of theinstrument, the bottom-most face of the pipette nozzles on the Z-payloadcan be capable of unobstructed travel in the Y direction from a point156 mm above the top surface of the baseplate to a point 256 mm abovethe top surface of the baseplate. The 1 ml pipette tips can be capableof penetrating the foil covers included on disposable regent strips.This penetration may not crate contamination, affect the associatedchemistries, or damage the pipette tips. Motions can be executed in sucha manner as to eliminate mechanical hysteresis, as needed. Gantrymotions can be optimized to prevent cross lane contamination andcarryover. The rack can align the reagent strips to a tolerance of+/=0.010 inches in the X and Y directions.

Referring now to the gantry, in some embodiments, the gantry can consistof a stepper-motor actuated, beltscrew-drive cartesian robotic system.The gantry can be flee to move, with or without attachments, above themodules that are forward of the rear facade and below the bottom-mosthorizontal face on the Z head, so long as the Z-payload is fullyretracted. The gantry can be capable of travel speeds up to about 500mm/sec in the X and Y directions and up to about 100 mm/sec in the Zdirection. The accuracy and precision of the axis motions (e.g., withrespect to the X, Y, and Z home sensors) can be 25 mm or better for eachaxis, and can be retained throughout the maintenance period. The axisdrive belts may not have residue in areas where PCR and samples areprocessed. The gantry can contain provisions for routing its own and allZ-payload wire harnesses back to the instrument. Belt tension on the Xand Y axes can be set at 41.5+/=3.5 pounds.

Referring now to the Z-payload, the fluid head can have 4 pipetteattachment nozzles located on 24 mm centers. Exemplary pipette tips thatthe pipette nozzles can capture without leakage include Biorobotix tipsPN23500048 (50 μL). PN23S00049 (1.75 μL), and PN23500046 (1 ml). The Zpayload can incorporate a stepper actuated stripper plate capable ofremoving pipette tips (e.g., the pipette tips described above). Thesystem can include a pump and manifold system that includes softwarecontrolled aspiration, dispensing, and venting of individual fluidvolumes within each of the four individual tips and simultaneousdispensing and venting on all tips. The pump and manifold system canhave an accuracy and precision of about +/=2 μL per tip for volumes thatare less than 20 μL and about +/=10% for volumes greater than or equalto 20 μL (e.g., when aspirating or dispensing in individual tips). Thetotal pump stroke volume can be greater than about 8 μL and less thanabout 1250 μL The minimum aspirate and dispense speed can be about 10μL/sec to about 300 μL/sec. The centroid of the bottom-most face of eachpipette tip can be axially aligned with the nozzle centroid of thepipette nozzles within 0.2 mm. The bottom-most pipette tip faces can beco-planar within 0.2 mm. The Z-payload can incorporate a Z axis forcesensor capable of feedback to software for applied forces of betweenabout 0 and 4 lbs. The Z-payload can incorporate a downward facingbarcode reader capable of reading the system barcodes as describedelsewhere herein.

Referring now to racks included in the system, disposable reagent strips(e.g., oriented orthogonally to the front of the instrument) can becontained in 2, 12-lane racks. The 12 regent strips in a given rack canregister and lock into the rack upon insertion by a user. The rack cancontain an area for 12 sample lysis tubes (e.g., PN 23500043) and holdthe tube bottoms co-planar, allowing the user to orient the bar code toface the rear of the instrument. Certain features, including thoselisted above, can allow the racks to be inserted and oriented in theinstrument by a minimally trained user. Proper rack placement can beconfirmed by feedback to the software. In some embodiments, the rackscan be black and color fast (e.g, the color may not appreciably degradewith use or washing with a 10% bleach solution) and the rack materialcan be dimensionally stable within 0.1 mm over the operating temperaturerange of the system. The rack can be designed with provisions to allowthe rack can be carried to end from the instrument and to minimize oreliminate the likelihood that the tubes held by the rack will spill whenplaced on a flat surface.

Referring now to the reader and PCR heater included in the system, thereader can allow for cartridge insertion and removal by, for example, aminimally trained user. The cartridge can remain seated in the readerduring system operation. In some embodiments, the cartridge barcode maynot be read properly by the barcode scanner if the cartridge is insertedincorrectly (e.g., upside down or backwards), thus the system caninstruct a user to correctly reinsert the cartridge into the reader traywhen the cartridge is inserted incorrectly. The reader drawer canrepeatably locate the cartridge, for loading by the pipette tips, within0.5 mm. The reader can deliver the cartridge from the loading positioninto a react and detect position by means of an automated drawermechanism under software control. The PCR lanes of the cartridge can bealigned, with both the optical system and heater, by the radar tray anddrawer mechanism. The cartridge can contact the beaters evenly withabout a 1 psi, or greater, average pressure in the areas of the PCRchannels and the wax valves. Heater wire bonds can be protected fromdamage so as not to interfere with system motion. Registration fromheater to cartridge and from cartridge to optical path centers can bewithin +/=0.010 inches. The reader can mechanically cycle a minimum ofabout 80,000 motions without failure.

Referring now to the one or more lysis heaters included in the system,the heaters for each of the 24 lysis stations can be individuallysoftware controlled. The lysis ramp times (e.g., the time that it takesfor the water in a lysis tube to rise from a temperature ofapproximately 2.5° C. to a given temperature) can be less than 120seconds for rise to 50° C. and less than 300 seconds for a rise to 75°C. The lysis temperature (e.g., as measured in the water contained in alysis tube) can be maintained, by the lysis haters, within +/=3° C. ofthe desired temperature. The accessible lysis temperature range can befrom about 40° C. to about 82° C. Each of the lysis heaters may drawabout 16 Watts or more of power when in operation. The lysis heater candesigned to maximize the thermal transfer to the lysis tube and alsoaccommodate the tolerances of the parts. The lysis heaters can permitthe lysis tubes to be in direct contact with the magnets (described inmore detail herein). The lysis beaters may be adjustable in thehorizontal plane during assembly and may not interfere with theinstalled covers of the system.

Referring now to magnets included in the system, the lysis and magnetrelated mechanisms can fit beneath the rack and may not interfere withrack insertion or registration. The magnets may be high-flux magnets(e.g., have about a 1,000 gauss, or greater, flux as measured within agiven lysis tube) and be able to move a distance sufficient to achievemagnetic bead separation in one or more of the lysis tubes filled to avolume of 900 μL. The magnets can be software-controllable at movementrates from about 1 mm/sec to about 25 m/see. The wiring, included aspart of the heater and controller assemblies, can be contained andprotected from potential spills (e.g., spills of the lysis tubes). Themagnets can be located about 1.25 inches or greater from the bottom ofthe lysis tube when not in use and can be restrained in such a manner asto maximize contact with the lysis tube while also preventing jamming.

In some embodiments, the system enclosure includes a semi-transparentlid (e.g., with opaque fixtures and/or hardware) in the front of theinstrument to allow users to view instrument functions. The lid caninclude a company and/or product logo and a graspable handle (e.g.,enabling the user to raise the lid). When closed, the lid can have anopening force no greater than 15 pounds (e.g., when measured tangentialto door rotation at the center of the bottom edge of the handle) and canlock in the open (e.g., “up”) position such that no more than about 5lbs. of force (e.g., applied at the handle and tangential to doorrotation) is required to overcome the handle lock and return the lid tothe closed position. The lid can include two safety lid locks that arenormally locked when power is not applied and can allow the system tomonitor the state (e.g., open or closed) of the lid. The lid can bedesigned such the lid does not fall when between the open and closedpositions. The enclosure can include a power switch located on the rightside of the instrument. A power cord can protrude from the enclosure insuch a way that positioning the instrument does not damage the cords orcause accidental disconnection. The enclosure can prevent the user fromcoming in contact with, for example, moving parts, high magnetic fields,live electrical connections, and the like. The enclosure can includefour supporting feet, located on the underside of the enclosure, toprovide a clearance of about 0.75 inches or more between the undersideof the enclosure and the table top. The enclose can include a recessedarea with access to external accessory connections such as the displayport, the Ethernet port, the 4 USB ports, and the like.

Referring now to the cooling sub-system included in the PCR system, anair intake can be provided in the front of the unit and an air exhaustcan be provided in the rear portion of the top of the unit. Intake aircan pass through the air intake and through a filter element (e.g., aremovable and washable filter element). The cooling sub-system canmaintain an interior air temperature (e.g., the temperature as ismeasured at the surface of the reagent strip, such as the reagent stripsnumbered 1, 12, and 24, at the surface of the PCR cartridges, and thelike) about 10° C. higher, or less than the ambient air temperature. Thecooling subsystem can maintain the internal air temperature at or belowabout 32° C. One or more cooling fins included as pat of the coolingsubsystem may require about 5.7 Watts, or less, of power per fan.

In some embodiments, the system can include covers on internalsubassemblies (with the exception of the gantry). The covers can becleanable with a 10% bleach solution applied with a soft cloth withoutsignificant degradation. The coven can supply a safety barrier between auser and the electronic and moving mechanical assemblies included in thesystem. The covers on the internal subassemblies can be designed tomaximize cooling of the internal subassemblies by maximizing airflowunder the covers and minimizing airflow above the covers. The covers canbe removable by a service technician and can match the color and textureof the enclosures.

In some embodiments, the system can be designed to operate within atemperature range of about 15° C. to about 30° C. and in anon-condensing relative humidity range (e.g., about 15% to about 80%relative humidity). The analyzer can be designed to perform withoutdamage after exposure to storage at no less than −20° C. for 24 hours orless, storage at no greater than 60° C. for 24 hours or less, and/orstorage at about 50,000 feet or less (e.g., 3.4 inches of Hg) for 24hours or less. The system can be designed with provisions to preventmotions that could damage the instrument during shipping. It can conformto the shipping standards set forth in ASTM D 4169-05, DC 12 and can bedesigned to allow the baseplate to be securely mounted to a shippingpallet. The racks and the enclosure of the instrument are designed notto degrade or be damaged by daily cleaning with a 10% bleach solution.The power to subassemblies of the system can be supplied by internalpower supplies. Exemplary power supplies can receive, as input, about1590 watts at about 90 to about 264 Vac at between about 47 and about 63Hz and supply about 1250 watts of output to the subassemblies.

In some embodiments, the system can include a power switch (e.g., arocker-type switch), located on the right side of the instrument, one ormore interface components, and/or one or more interface ports. Forexample, the system can include an LCD display monitor that is 15inches, has 1280×1024 pixel resolution and 16-bit color. The system canalso include other display monitors such as ones with increased size,resolution, and/or color depth. The LCD display can be connected to thesystem via a VGA connection. The system an include a while, 2 button USBmouse, a white USB keyboard, a black SIT power cable, and anun-interruptile power supply, with feedback through USB. The system canalso include a USB color printer, 2 USB cables (e.g., one for theprinter and one for the UPS). The system can include exemplary interfaceports, such as, 4 USB ports (e.g., to connect to a pointing device,printer, keyboard, UPS, LIS), 1 VGA port (e.g., for connection to theLCD display), and 1 Ethernet port (e.g., for PC connectivity) located onthe left side of the enclosure. An IEC/EN 60320-11C14 power port can beincluded n the right side of the enclosure.

In some embodiments, the system can include features directed atincreasing the safety of a user. For example, door interlocks can beincluded to prevent user access while the gantry is in motion and/orwhile other non-interruptible processes are underway. The system an bedesigned to minimize or eliminate the presence of user-accessibledangerous corners and/or edges on the instrument and designed such thatmetal part are properly electrically grounded. Sheet metal or plasticcovers can be included over mechanical and electrical components asnecessary to protect a user from moving parts and/or live electricalparts and to protect the electronics and motors included in the systemtom, for example, spills.

Example 19: Exemplary Optics

Described herein are exemplary specifications related to the design ofoptics used in a PCR Analyzer and/or System. Additional informationrelated to the PCR System is described elsewhere herein. The opticaldetection system included in the PCR System can be a 12-lane two-colordetection system for monitoring real-time PCR florescence from a 12-lanemicrofluidic PCR cartridge. The system can include excitation lights(e.g., blue and amber LED light sources), one or more band pass fillers,and one or more focusing leases. The emitted fluorescence light from thePCR reactor (e.g., included in the microfluidic cartridge) is capturedthough a pathway into a focusing lens, through a filter, and onto aphotodiode. Included in the system, for each PCR lane, are dedicated,fixed individual optical elements for each of the two colorsinterrogated.

In some embodiments, the limit of detection is 20 DNA copies perreaction of input PCR reaction mix with a minimum signal to base valueof 1.1 S. The 2 color fluorescence system can be used with, for example,FAM (or equivalent) and Cal Red (or equivalent). The system can have theability to collect fluorescence data in about 100 ms to about 600 ms atthe maximum rate of one data point every about two seconds. Whencollecting data from a PCR lane, LEDs in adjacent lanes increase thesignal in the lane being sampled by less than about 1% (e.g., 0.5%). Thenoise of the detection can be less than about 1% of the maximum signal.The lane-to-lane fluorescence variability with a fluorescence standard(e.g., part #14000009) can be within Cv of 30% for both FAM and Cal Red,when measured using the dark current-corrected-fluorescence-slope. Theaverage dark current-corrected-fluorescence-slope for the optical blockwith 12 lanes can be between about 30 mV to about 90 mV/(% blue LEDpower) for FAM using the fluorescence standard (Part #14000009). Theaverage dark current-corrected-fluorescence lope for the optical blockwith 12 lanes should be between about 75 mV to about 300 mV/(% amber LEDpower) for Cal Red using the standard fluorescence cartridge (Part14000009). The average excitation power for each channel can beindependently varied by software from about 5% to about 100%. There maybe no source of light activated inside the reader to affect thefluorescence reading. In some embodiments, turning room lights on or offdoes not affect the optical readings.

In some embodiments, the system can include an optical block with 12repeats of 2-color fluorescence detection units at a pitch of about 8mm. The optical detection block can be positioned on top of themicrofluidic cartridge, with excitation and emission travelling throughthe PCR windows of the microfluidic cartridge. The apertures of theoptical block an align with the PCR reactor within about +/=200 microns.An optical electronics board containing the LEDs and Photodetectors canbe mated flush with the top of the optics block with each of thephotodetector recessed into the bores of its corresponding optical lane.When the microfluidic cartridge is installed in the system, the opticalblock can be used to deliver a force of about 20 to about 30 lbs. overthe active area of the microfluidic cartridge with an average pressureof at least about 1 psi.

The optical block can be made of aluminum and surfaces present in theoptical path lengths can be anodized black, for example, to minimizeauto-fluorescence a well a light scattering. An aperture plate having 12slits, each slit about 10 mm in length and 1 mm wide, can be used, forexample to limit the size of the excitation light spots as well asreduce background fluorescence. The thickness of the optics block can beabout 1.135+/=0.005 inches. The bottom surface of the optics block canbe planar within +/=1 mil to provide uniform pressure over the microfluidic cartridge. The apertures should be kept clean and free of debrisduring manufacturing of the optics block and assembly of the opticsblock into the system.

In some embodiments, the system can include excitation optics with anangle of excitation path equal to 55+/=0.5 inches with respect to normalof the PCR cartridge surface. One exemplary arrangement of opticalelements in the excitation path, in order, is LED, lens, filter,aperture, and PCR sample. The system can use a Planoconvex excitationlens (e.g., PCX, 6×9, MgF2TS) oriented with the flat side toward the PCRsample. Included in the optics are one or more excitation paths withtapers that can be designed such that the lens and filter an be placedinside the bore to provide a light spot bigger than the aperture plate.The location of the LED and the sample can be fixed as the design caninclude a fixed available optical block thickness. The location of thelens and the filter can be determined to provide a excitation spot sizeof about 6 mm along the length of a PCR lane. The excitation optics caninclude a LED such as Luxeon Part # LXK2-PB 14-N00 (e.g., for FAMexcitation) that includes a center wavelength of about 470 nm (blue)with a half band width of about 75 nanometers, or less (e.g., for FAMexcitation). The excitation optics can also include an LED such asLuxeon Part U LXK2-PL12-Q00 (e.g., for Cal Red excitation) that includesa center wavelength of 575 nm (amber) with a half band width of about 75nanometers, or less (e.g., for Cal Red excitation). The LEDs used in theexcitation optics can remain stable for about 5 years or more or about10,000 cycles.

The system can include emission optics with an angle of emission pathequal to about 15+/=0.5 inches with respect to normal of the PCRcartridge surface. One exemplary arrangement of optical elements in theemission path, in order, is PCR sample, aperture, filter, lens, andphotodetector. The emission lens can be plan-convex (e.g., PCX, 6×6MgF2TS) with the flat side toward the photodetectors. The emissionoptics can include one or more bores, for the emission path, with tapersthat can be designed so as to maximize detected light while enablingsnug placement of the filers and lenses. The location of thephotodetectors with respect to the sample can be fixed as the design caninclude a fixed available optical block thickness. The location of thelens and the filter can be determined so as to provide an emission spotsize of 6 mm along the length of a PCR lane. An exemplary photodetectorthat can be used in the emission optics is the Hamamatsu SiliconPhotodetector with Lens, S2386-18L.

In some embodiments, the system can include one or more filters withdiameters of about 6.0+/=0.1 mm, thicknesses of about 6.0+/=0.1 mm,clear apertures with diameters of less than or equal to about 4 mm. Thefilters can include a blackened edge treatment performed prior toplacement in a mounting ring. If present, the mounting ring can be mealand anodized black. The filter can be manufactured from optical glasswith a surface quality that comprises with F/F per Mil-C-48497A, an AOIof about 0 dog, a 12 cone AOI of about +8 deg, and ca be humidity andtemperature stable within the recommend operating range of the system.An exemplary filter can be obtained from Omega Optical Brattleboro, Vt.05301.

The system can include one or more FITC Exciter Filters (e.g., PN14000001) with an Omega pert number 481 AF30-RED-EXC (e.g., drawing#2006662) used, for example, in FAM excitation. These filters can have acut-on wavelength of about 466+/=4 nm and a cut-off wavelength of about496+0/=4 nm. The transmission of filters of this type can be greaterthan or equal to about 65% of peak. These filters can have a blockingefficiency of greater than or equal to OD4 for wavelengths ofultraviolet to about 439 nm, of greater than or equal to OD4 forwavelengths of about 651 nm to about 1000 nm, of greater than or equalto OD5 for wavelengths of about 501 nm to about 650 nm, and of greaterthan or equal to OD8, in theory, for wavelengths of about 503 nm toabout 580 nm.

The system can include one or more Amber Exciter Filters (e.g., PN14000002) with a part number 582AF25-RED-EXC (e.g., drawing #2006664)used, for example, in Cal Red excitation. These filters can have acut-on wavelength of about 569+/=5 nm and a cut-off wavelength of about594+0/=5 nm. The transmission of filers of this type can be greater thanor equal to about 70% of peak. These filters can have a blockingefficiency of reactor than or equal to OD8, in theory, for wavelengthsof about 600 nm to about 700 nm.

The system can include one or more FITC Emitter Filters (e.g., PN14000005) with a part number S34AF40-RED-EM (e.g., drawing #2006663)used, for example, in FAM emission. These filters can have a cut-onwavelength of 514+/=2 nm and a cut-off wavelength of 554+/=5 nm. Thetransmission of filters of this type can be greater than or equal toabout 70% of peak. These filters can have a blocking efficiency ofgreater than or equal to OD5 for wavelengths from ultraviolet to about507 nm of greater than or equal to OD8, in theory, from about 400 nm toabout 504 nm, and of greater than or equal to OD4 avg, from about 593 nmto about 765 nm.

The system can include one or more Amber Emitter Filters (e.g., PN14000006) with a part number 627AF30-RED-EM (e.g., drawing #2006665)used, for example, in Cal Red emission. These filters can have a cut-onwavelength of 612+5/=0 nm and a cut-off wavelength of 642+/=nm. Thetransmission of filters of this type can be greater than or equal toabout 70% of peak. These filters can have a blocking efficiency ofgreater than or equal to OD5 for wavelengths from ultraviolet to about605 nm, of greater than or equal to OD8, in theory, from about 550 nm toabout 600 nm, and of greater than or equal to OD5 avg, from about 667 nmto about 900 nm.

Example 20: Exemplary 3-Layer Cartridge

Described herein are exemplary specifications used to design andassemble the microfluidic cartridge as well as exemplary instructions onthe use of the cartridge in, for example, the system described herein.In some embodiments, the cartridge can have a maximum limit of detectionequal to 20 copies per reaction volume (e.g., 20 copies/4μ), with atarget detection of 10 copies per reaction volume. The cartridge canperform 45 reaction cycles in 40 minutes or less (e.g., 45 cycles in 40minutes, 45 cycles in 20 minutes, 45 cycles in 15 minutes, or the like).The cartridge can utilize two color detection using, for example, theFAM (or equivalent) and CAL RED (or equivalent) fluorescent dyes.Results obtained using the cartridge have been compared with the resultsobtained using standard real-time PCR instruments.

In some embodiments, the Cartridge can be a one-time use, disposablecartridge that can be disposed of according to typical laboratoryprocedures. The cartridge can be 4.375 inches long and 2.800 incheswide, with a thickness of 0.094+/=0.005 inches. The cartridge caninclude features that allow the cartridge to interface with, forexample, the system described herein. Exemplary interfacing featuresinclude PCR channel walls and the top of the micro-substrate over thePCR channel that are well polished (SPI A1/A2/A3), enabling easytransfer of excitation and emission light between the PCR reactor (e.g.,contained in the cartridge) and the detection system (e.g., theanalyzer). The cartridge can include a thermal interface, located on thebottom of the cartridge, for interfacing with the analyzer. The thermalinterface can have a thin laminate (e.g., less than 150 microns thick,100 microns thick, or the like) to encourage heat transfer from theheater wafer to, for example, the PCR channels of the cartridge.

The cartridge can include one or more mechanical interfaces with, forexample, the analyzer. For example, the cartridge can have a notch inone or more of the corners that can mate with a corresponding shape onthe heater module of the analyzer. The notch and corresponding shape canenable the cartridge to be placed only one way in the tray of, forexample, the system described herein. In some embodiments, the cartridgehas a single notch in one of the corners, with the remaining threecorners having a minimum radius of 1 mm to facilitate placement of thecartridge in the analyzer. During use (e.g., when placed in a systemdescribed herein and performing a function such as PCR), the cartridgecan be pressed, on one side, by the optics block, against the heaterwafer (positioned against the opposite side), with a pressure of about 1psi or greater (e.g., 0.99 psi, 1.2 psi, or the like). When located inthe tray of the analyzer, the cartridge can have an alignment slop of+/=200 microns to enable a user to easily place and remove the cartridgefrom the analyzer tray. The cartridge can have two ledges, that are each1 mm wide and located along the two long edges of the cartridge, toenable the heating surface to extend below the datum of the tray.

In some embodiments, the cartridge can have the following functionalspecifications. The cartridge can include an inlet hole that is, forexample, cone-shaped with a height of 1 mm from the top surface of thecartridge. The cone can have an inner diameter of 3 mm at the top of thecone and can taper down to a diameter that matches the width of amicrochannel (e.g., an inlet channel) that the inlet cone is fluidlyconnected to. The inlet channel can fluidly connect the inlet hole to aPCR reactor that has an interior volume of, for example, about 4.25 μlto 4.75 μl (e.g., 4.22 μl, 4.5 μl, 4.75 μl, or the like). An outletmicrofluidic channel can fluidly connect the PCR reactor to an overflowchamber. The cartridge can also include an outlet vent hole.

The input PCR sample (e.g., a reaction mixture) can be between about 6.0and 7.0 μl per PCR lane (e.g., 5.9 μl per lane, 6.4 μl per lane, 7.1 μlper lane, or the like) and can be introduced into the cartridge throughthe inlet hole by, for example, a pipette. The reaction mixture can betransported, via the inlet channel, to the PCR reactor where thereaction mixture can be isolated (e.g., sealed off by valves) to preventevaporation or movement of the reaction mixture during thermocycling.Once the mixture is sealed inside the chamber, the analyzer can initiatemultiplexed real-time PCR on some or all of the reaction mixture (e.g.,4.5 μl, an amount of fluid equal to the inner volume of the reactionchamber, or the like).

The microfluidic substrate of the cartridge can include one or more ofthe following specifications. The material of the microsubstrate can beoptically clear (e.g., have about 90% or greater optical transmission,be 3 mm thick, comply with ASTMD1003, and the like), haveauto-fluorescence that is less than that emitted by 2 mm thick ZEONOR1420R, and have a refractive index of about 1.53 (ASTM D542). Thematerial of the microsubstrate can be amenable to the injection moldingof features required for the microfluidic network of the cartridge. Thematerial is preferably compatible with all PCR agents and can withstandtemperatures of up to about 130° C. for about 5 minutes or more withoutyielding or melting. The cartridge can include fiducials, recognizableby HandyLab manufacturing equipment, located in one or more (preferablytwo) of the corners of the substrate. The cartridge can include fluidiccomponents (e.g., microchannels, valves, end vents, reagent inlet holes,reaction chambers, and the like) necessary to perform the functions ofthe cartridge (e.g., PCR).

Additional features of the substrate material can include one or more ofthe following. Minimum clearances of about 1 mm can be designed betweenfunctional features to ensure sealing success (e.g., to the analyzer),and to allow simplified fixturing during assembly. The cartridge caninclude dogbones under small fluid path ends to, for example, increasemold life. The bottom of the micro tool surface can be roughened (e.g.,by vapor hone, EDM, or the like). The substrate material can be capableof adhesion by a label.

In some embodiments, the sealing tape used in the cartridge can includeone or more of the following specifications. Laminate can be easilyapplied to the bottom of the microfluidic substrate. Material of thelaminate is preferably pin-hole free. The material and adhesive ispreferably compatible with the PCR reaction chemistries. The laminatematerial and glue used should not auto-fluoresce. The material canwithstand up to 130° C. for 5 minutes without losing adhesion, yielding,melting, or causing undue stresses on the cartridge. Bubbles should notform in the adhesive layer upon heating (e.g., to 130° C. for 5 minutes)after application to the microsubstrate. The laminate should be lessthan 5 mills thick to, for example, enable rapid heat transfer.

The high temperature wax included in the cartridge can have thefollowing characteristics. The wax should have a melt point of about90+/=3° C. (e.g., 87° C., 90° C., 93.1° C., or the like), bebiocompatible with PCR reactions, have wettability with microsubstratematerial, and have a melt viscosity range, for example, of aboutViscosity at 100° C.-20 mm²'s and Hardness at 25° C.=8 dmm. The mainlabel of the cartridge can have the following characteristics. It canhave a thickness of 2-4 mils, have suitable bondability to microfeatures and seal around the valves, include cuts for one or more PCRwindows, and a tab (free from adhesive) for aiding in removal of thecartridge from the analyzer. The main label can also have abrasionresistance on the top surface, and be printable. The main label can havean upper and lower alignment pattern for the label to completely coverthe valve holes for proper operation of the valves.

The cartridge can include a barcode label applied to the top of thecartridge that is readable by a barcode reader (e.g., the barcode readerincluded in the analyzer) while the cartridge is installed in theanalyzer. The barcode label can include the product name, lot 9,expiration date, bar code (2D) and may be printed on. In addition, or inthe alternative, a barcode may be applied directly to the main cartridgelabel using a laser or inkjet type printer.

The packaging that the cartridge is included in can include one or moreof the following: package label, carton, carton label, and/or operatinginstructions. The packaging can be printed on or label attachable,placed inside of a plastic bag, shrink/stretch wrap bag, or the like,and can be stacked in groups of 24. The cartridge bagging without acritical seal should be kept free from dust contamination.

The cartridge can include one or more valves (e.g., temperaturecontrolled, wax-containing valves) for starting, stopping, and/orcontrolling the flow of material inside the cartridge. The wax containedin the valves can be free of trapped air bubbles that have a diametergreater than half the width of the valve channel. The valve channel canhave an air pocket. The wax may not intrude into the fluid path prior toactivation. The wax can be filled to the start of the flare to the fluidpath.

The cartridge can include micro channels and holes such that the holesare of a size and shape to enable easy, leak-free interfacing with a 175μl pipette tip. In some examples, the holes size is between about 200 μmand about 4000 μm in diameter. The microchannels can be between about 50μm and about 1500 μm wide and between about 50 μm and 1000 μm high.

The cartridge can include valves for controlling the flow of fluidwithin the cartridge (e.g., through the microchannels, reactor chambers,and the like). The valve edges, steps, and general geometry can bedesigned to encourage exact flow and/or stoppage required during waxload. The valve geometry can be designed to accommodate limitations ofwax dispensing equipment (e.g., =1-25% of 75 nL volume). In someembodiments, step down air chambers on the valves are funnel shaped toaid wax loading and the remaining geometry diminishes from the bottom ofthe funnel to the end point where the wax stops. The path where thevalves are to flow into and block, during use, can be narrow enough(e.g., 150-200 microns wide and deep) and have enough length toeffectively seal when the valves are activated during use. The valve waxtemperature can be about 90° C. When in use to block a portion of amicrochannel, the valves can seal to prevent evaporation of fluid and/orphysical migration of fluid from the PCR reactor during thermocycling.

The cartridge can include one or more PCR regions for performing PCR ona sample. The channel in the PCR region (e.g., PCR reactor) can bedesigned such that the temperature of the contents of the channel remainuniformly within about 1° C. of the anneal temperature. The channelwalls can have a polish of SPI A1/A2/A3.

In some embodiments, the cartridge is designed to be able to performdiagnostic tests within a temperature range of about 59° F. to about 86°F. (about 15° C. to about 30° C.) and a humidity range of about 15%relative humidity to about 80% relative humidity. The cartridge isdesigned to be safe and functional when used indoors, used at analtitude of 2000 m or less, and used under non-condensing humidityconditions (e.g., maximum relative humidity of 80% for temperatures upto 31° C. decreasing linearly to 50% relative humidity at 40° C.).

In use, PCR product produced in the cartridge can remain in the usedcartridge to, for example, minimize the likelihood of crosscontamination. The cartridge can be designed such that a 4 foot drop ofthe cartridge, while in its packaging, will not damage the cartridge.The cartridge is designed to perform without damage ater exposure to thefollowing conditions. The cartridge should be stored at 4° C. to 40° C.for the rated shelf life. Exposure to temperatures between −20° C. and4° C. or 40° C. and 60° C. should occur for no longer than 24 hours. Thecartridge can withstand air pressure changes typical of air transport.

The cartridge can be labeled with the following information (e.g., toidentify the cartridge, comply with regulations, and the like). Thelabel can contain a “Research Use Only” label, if applicable, and a CEmark, if applicable. The label can contain the company name and logo(e.g., Handylab), a part number (e.g., 55000009), a part name (12×Cartridge-nonvented), a lot number (e.g., LOT 123456), an expirationdate (e.g., June 2015), space for writing, a barcode according tobarcode specifications (described elsewhere), and/or “Handylab, Inc. AnnArbor, Mich. 48108 USA”.

The cartridge can be include in a carton that can contain informationsuch as, a part number (e.g., 55000009), a part name (12×Cartridge-nonvented), a quantity (e.g., 24), a lot number (e.g., LOT123456), an expiration date (e.g., June 2015), an optional UPC code,“Manufactured by Handylab, Inc., Ann Arbor, Mich. 48108 USA”, a cartonlabel to state storage limits, a CE mark (if applicable), and/or an ARname and address.

The cartridge packaging can include paper wrap to secure multiplecartridges together and clean package fill to prevent damage, forexample, from vibration. The cartridge shipping carton can includefeatures such as, compliance to ASTM 6159, carton may be stored in anydirection, refrigeration or fragile labeling of the carton may not berequired, and additional cold packs may not be required. The shelf lifeof the cartridge is 12 months or more.

The cartridge can comply with IEC 61010 (NRTL tested) and an FDA listingmay be required for clinical distribution. Cartridges used in a clinicallab device may meet all quality system requirements. Cartridges used forresearch only in a commercial device may meet all HandyLab qualitysystem requirements. Cartridges for research use only (Alpha or Betatesting) may be design/manufacturing traceable to a DHR (manufacturingrecord).

The foregoing description is intended to illustrate various aspects ofthe present inventions. It is not intended that the examples presentedherein limit the scope of the present inventions. The technology nowbeing fully described, it will be apparent to one of ordinary skill inthe art that many changes and modifications can be made thereto withoutdeparting from the spirit or scope of the appended claims.

1. (canceled)
 2. A system for analyzing a plurality of nucleicacid-containing samples, the system comprising: a first moduleconfigured to extract nucleic acids from the plurality of nucleicacid-containing samples, a second module configured to simultaneouslyamplify the nucleic acid extracted from the plurality of nucleicacid-containing samples, the first and second modules comprising: a bayconfigured to removably receive a housing comprising a plurality ofprocess chambers that are maintained at a same height relative to oneanother as the housing is received in and removed from the bay, theplurality of process chambers aligned along a first axis when thehousing is received in the bay, the bay comprising one or morecomplementary registration members configured to receive the housing ina single orientation when the housing is received in the bay, the firstmodule further comprising a magnetic separator positioned to apply amagnetic force to a first side of the plurality of process chambers whenthe housing is received in the bay, the magnetic separator comprisingone or more magnets aligned along a second axis parallel to the firstaxis when the housing is received in the bay, the one or morecomplementary registration members further configured to align theplurality of process chambers with the magnetic separator when thehousing is received in the bay, and the first module further comprisinga heating assembly positioned adjacent to a second side of the pluralityof process chambers opposite the first side when the housing is receivedin the bay, the heater assembly comprising one or more heaters alignedalong a third axis parallel to the first axis when the housing isreceived in the bay, the heating assembly configured to heat a solutionin the plurality of process chambers to between 50° C. and 85° C., theone or more complementary registration members configured to align theplurality of process chambers with the heater assembly when the housingis received in the bay; and a liquid dispenser configured to movebetween the plurality of nucleic acid-containing samples and theplurality of process chambers when the housing is received in the bay,the liquid dispenser configured to dispense at least a portion of theplurality of nucleic acid-containing samples and a plurality of magneticbinding particles into the plurality of process chambers when thehousing is received in the bay, the liquid dispenser further configuredto dispense the nucleic acid extracted from the plurality ofnucleic-acid containing samples into the second module.
 3. The system ofclaim 2, wherein the plurality of nucleic acid-containing samples are inone-to-one correspondence with the plurality of process chambers whenthe housing is received in the bay.
 4. The system of claim 2, whereinthe liquid dispenser comprises one or more dispense heads configured toaccept a pipette tip.
 5. The system of claim 4, wherein the liquiddispenser comprises four dispense heads and the housing comprises twelveprocess chambers, each dispense head configured to dispense a pluralityof magnetic binding particles and at least a portion of one sample ofthe plurality of nucleic acid-containing samples into one of the twelveprocess chambers when the housing is received in the bay.
 6. The systemof claim 2, further comprising a sample identification verifierconfigured to check an identity of each sample of the plurality ofnucleic acid-containing samples, wherein the sample identificationverifier is selected from the group consisting of an optical characterreader, a bar code reader, and a radio frequency tag reader.
 7. Thesystem of claim 2, further comprising electronic circuitry configured tocontrol operation of the magnetic separator, the heater assembly, andthe liquid dispenser.
 8. The system of claim 7, wherein the electroniccircuitry is configured to cause the magnetic separator to apply amagnetic force to the plurality of process chambers when the housing isreceived in the bay.
 9. The system of claim 7, wherein the electroniccircuitry is configured to cause the heater assembly to apply heat tothe plurality of process chambers when the housing is received in thebay.
 10. The system of claim 7, wherein the electronic circuitry isconfigured to control motion of the liquid dispenser when the housing isreceived in the bay.
 11. The system of claim 2, further comprising oneor more processors and at least one input device coupled to the one ormore processors, the at least one input device selected from the groupconsisting of: a keyboard, a touch-sensitive surface configured toaccept input from a stylus or a user's finger, a microphone, atrack-pad, a retinal scanner, a fingerprint reader, a holographicallyprojected interface, and a mouse.
 12. The system of claim 11, furthercomprising a communication interface coupled to the one or moreprocessors, the communication interface being selected from the groupconsisting of: a serial connection, a parallel connection, a wirelessnetwork connection, a wired network connection, and one or more USBports.
 13. The system of claim 12, further comprising a data storagemedium configured to receive data from the one or more processors, theat least one input device, and the communication interface, the storagemedium being selected from the group consisting of: a hard disk drive,an optical disk drive, a flash-card, a USB-drive, and a CD-Rom.
 14. Thesystem of claim 13, further comprising at least one output devicecoupled to the one or more processors, the at least one output devicebeing selected from a visual display, a printer, a holographicprojection, and a speaker.
 15. The system of claim 2, further comprisingmore than one bay, each bay configured to removably receive a housingcomprising a plurality of process chambers.
 16. The system of claim 2,wherein the number of nucleic acid-containing samples is twelve.
 17. Thesystem of claim 2, further comprising an optical detection systemconfigured to independently detect a plurality of fluorescent dyes at aplurality of different locations, wherein each fluorescent dye binds toa fluorescent polynucleotide probe or a fragment thereof.
 18. The systemof claim 17, wherein the optical detection system selectively emitslight in an absorption band of the plurality of fluorescent dyes andselectively detects light in an emission band of the plurality offluorescent dyes.
 19. The system of claim 17, configured to carry outextraction, amplification, and detection of the plurality of nucleicacid-containing samples in less than an hour.
 20. The system of claim 2,further comprising a heater substrate comprising at least one heatsource configured to apply heat at one or more selected times in orderto apply thermocycling operations sufficient to amplify the nucleic acidextracted from the plurality of nucleic acid-containing samples.
 21. Thesystem of claim 20, wherein the at least one heat source is configuredto maintain a negligible temperature gradient across a reaction zoneduring the thermocycling operations, the reaction zone configured toreceive the nucleic acid extracted from one of the plurality of nucleicacid-containing samples.
 22. The system of claim 21, wherein the atleast one heat source is configured to maintain a negligible temperaturegradient across each of a plurality of reaction zones during thethermocycling operations, each reaction zone configured to receive thenucleic acid extracted from one of the plurality of nucleicacid-containing samples.
 23. A system for analyzing a plurality ofnucleic acid-containing samples, the system comprising: a first moduleconfigured to extract nucleic acids from the plurality of nucleicacid-containing samples, the first module comprising: a bay configuredto removably receive a housing comprising a plurality of processchambers that are maintained at a same height relative to one another asthe housing is received in and removed from the bay, the plurality ofprocess chambers aligned along a first axis when the housing is receivedin the bay, the bay comprising one or more complementary registrationmembers configured to receive the housing in a single orientation whenthe housing is received in the bay, a magnetic separator positioned toapply a magnetic force to a first side of the plurality of processchambers when the housing is received in the bay, the magnetic separatorcomprising one or more magnets aligned along a second axis parallel tothe first axis when the housing is received in the bay, the one or morecomplementary registration members further configured to align theplurality of process chambers with the magnetic separator when thehousing is received in the bay, and a heating assembly positionedadjacent to a second side of the plurality of process chambers oppositethe first side when the housing is received in the bay, the heaterassembly comprising one or more heaters aligned along a third axisparallel to the first axis when the housing is received in the bay, theheating assembly configured to heat a solution in the plurality ofprocess chambers to between 50° C. and 85° C., the one or morecomplementary registration members configured to align the plurality ofprocess chambers with the heater assembly when the housing is receivedin the bay; a second module configured to receive a multi-lanemicrofluidic cartridge configured to simultaneously amplify the nucleicacid extracted from the plurality of nucleic acid-containing samples;and a liquid dispenser configured to move between the plurality ofnucleic acid-containing samples and the plurality of process chamberswhen the housing is received in the bay, the liquid dispenser configuredto dispense at least a portion of the plurality of nucleicacid-containing samples and a plurality of magnetic binding particlesinto the plurality of process chambers when the housing is received inthe bay, the liquid dispenser further configured to dispense the nucleicacid extracted from the plurality of nucleic-acid containing samplesinto the multi-lane microfluidic cartridge.
 24. The system of claim 23,wherein the liquid dispenser comprises one or more dispense headsconfigured to accept a pipette tip.
 25. The system of claim 24, whereinthe liquid dispenser comprises four dispense heads and the housingcomprises twelve process chambers, each dispense head configured todispense a plurality of magnetic binding particles and at least aportion of one sample of the plurality of nucleic acid-containingsamples into one of the twelve process chambers when the housing isreceived in the bay.
 26. The system of claim 23, further comprisingelectronic circuitry configured to control operation of the magneticseparator, the heater assembly, and the liquid dispenser.
 27. The systemof claim 23, further comprising one or more processors and at least oneinput device coupled to the one or more processors, the at least oneinput device selected from the group consisting of: a keyboard, atouch-sensitive surface configured to accept input from a stylus or auser's finger, a microphone, a track-pad, a retinal scanner, afingerprint reader, a holographically projected interface, and a mouse.28. The system of claim 23, wherein the first module comprises more thanone bay, each bay configured to removably receive a housing comprising aplurality of process chambers.
 29. The system of claim 23, wherein thenumber of nucleic acid-containing samples is twelve.
 30. The system ofclaim 23, further comprising an optical detection system configured toindependently detect a plurality of fluorescent dyes at a plurality ofdifferent locations, wherein each fluorescent dye binds to a fluorescentpolynucleotide probe or a fragment thereof.
 31. The system of claim 23,further comprising a heater substrate comprising at least one heatsource configured to apply heat at one or more selected times in orderto apply thermocycling operations sufficient to amplify the nucleic acidextracted from the plurality of nucleic acid-containing samples.